JP3312867B2 - Method for transforming plant and method for producing transformed plant - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently transforming a plant and a method for producing a transformed plant.

[0002]

2. Description of the Related Art There have been known several attempts to transform a plant cell by introducing a gene into the cell to obtain a transformed plant.

[0003] For example, a method of using a whisker such as silicon carbide fiber to mix plant cells vigorously under liquid mixing, perforating by this operation, and introducing a foreign gene (hereinafter abbreviated as "whisker method") ( U.S. Pat. No. 5,302,523, U.S. Pat. No. 7,472,538, Kaepper, HF et al., 1992,
"Theoretic and Applied
Genetics ", Vol. 84, pp. 560-566, A
Sano, Y. et al., 1991, "Plant Scie".
nce, Vol. 79, pp. 247-252), a method of irradiating plant cells with ultrasonic waves to partially damage the cells, and introducing a gene through the damaged site (Joersbo, M., et al.
Et al., 1990, "Plant Cell Reporter
ts ", Vol. 9, pp. 207-210, Joersbo,
M. Et al., 1992, "Physiological Pl.
antarum ", Vol. 85, pp. 230-234), but a transformed plant has not been efficiently obtained by any of these methods.

[0004]

The conventional whisker method or ultrasonic method for gene transfer makes it difficult to obtain a transformant and is not a practical method for transforming plants.

[0005] Therefore, it is desired to develop a method for efficiently introducing a gene into dicotyledonous and monocotyledonous plant cells to obtain a transformed plant.

[0006] It is an object of the present invention to solve the above problems and to provide a method for transforming a plant and a method for producing a transformed plant, which have a high transformation rate.

[0007]

Means for Solving the Problems The present inventors have intensively studied to achieve these objects. As a result, in a state where the plant cells or plant tissues are dispersed in a liquid together with the foreign gene and whisker, the foreign gene is introduced into the cells by performing centrifugation and ultrasonic treatment, whereby the trait in which the foreign gene is expressed is obtained. It has been found that transformed cells can be obtained by efficiently obtaining transformed cells and regenerating them.

That is, the gist of the first invention is a method for transforming a plant, which comprises the following steps (a) to (c). (A) dispersing a plant cell or a tissue containing a plant cell in a liquid together with a foreign gene and whisker;
After the step (a), a centrifugal treatment is performed to attach whiskers to the plant cells. (C) After the step (b), an ultrasonic treatment is performed to pierce the plant cells with the whiskers. A step of introducing a foreign gene into the cell.

[0009] The gist of the second invention is a method for producing a transformed plant, which comprises the following steps (a) to (d). (A) dispersing a plant cell or a tissue containing a plant cell in a liquid together with a foreign gene and whisker;
After the step (a), a centrifugal treatment is performed to attach whiskers to the plant cells. (C) After the step (b), an ultrasonic treatment is performed to pierce the plant cells with the whiskers. A step of introducing a foreign gene into the cell. (D) a step of regenerating a plant from a plant cell into which the foreign gene has been introduced in step (c) or a tissue containing the same.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The method of the present invention will be described below in detail. The method for transforming a plant of the present invention comprises the steps (a) to
The method for producing a transformed plant of the present invention, which comprises the steps (c) and (c), further comprises the step (d) added to the above steps.

Hereinafter, (a), (b), (c) and (d)
Each step will be described in detail.

[1] Step (a) Dispersion Step The present invention is a step of uniformly dispersing a plant cell or plant tissue in a liquid together with an exogenous gene and whisker to make a suspended state.

The plant to which the method of the present invention is applied is not particularly limited. For example, rice, corn, wheat, barley, turfgrass, other monocotyledonous plants, or
Tobacco, soybean, cotton, tomato, Chinese cabbage, cucumber,
Examples include dicotyledonous plants such as lettuce.

The plant cells used in the present invention include, for example, dedifferentiated cultured cells such as callus and suspension cells, adventitious embryos, shoot primordia, and plant tissues such as leaves, roots,
Examples include a stem, an embryo, a growth point, and the like, and preferred are plant cells such as callus and suspension cells.

The cultured cells used in the present invention may be any explants derived from plants, for example, scutellum,
Growth points, pollen, anthers, leaf blades, stems, petiole, roots and the like can be mentioned.

The cultured cells used in the present invention may be obtained by using the above-mentioned explants in a callus forming medium, for example, an MS medium (Mu medium).
Rasige et al., Physilogia Pla.
ntarum ", 1962, Vol. 15, pp. 473-4
97) or R2 medium (Ojima et al., “Plan
t and Cell Physiology ", 19
73, Vol. 11, 1113-1121, N6 medium (Chu et al., 1978, "In Proc. Sym."
p. Plant Tissue Culture, Sc
issue Press Peking, ”p. 43-5
0), etc., in a medium containing an inorganic salt component and pitamines as essential components.
-PA (2,4-dichlorophenoxyac)
eticacid) 0.1 to 5 mg / liter, and as a carbon source, for example, sucrose 10 to 60 g / liter and gellite 1 to 5 g / liter.

After the explant is placed on the callus formation medium, the culture period until the cultured cells used in the present invention are obtained is not particularly limited. It is important that the plant can be regenerated from the cultured cells, that is, it is important that the plant cells have a plant regeneration ability.

The cultured cells used in the present invention may be suspension cells in a liquid medium as long as the cells have a plant regeneration ability.

The whisker used in the present invention refers to a single crystal having a fine needle-like structure. The whisker used in the present invention is not limited as long as it can stab cells, penetrate the cell wall, form pores on the cell surface, and introduce foreign genes into the cells via these pores. Although not critical, the pore size should not be too large compared to the cell size. Specifically, the diameter is 0.01 to 10 μm, preferably 0.1 to 10 μm.
5 to 1 μm, length is 1 to 100 μm, preferably 3 to
40 μm. Whisker material is potassium titanate,
Calcium carbonate, aluminum borate, silicon nitride, zinc oxide, basic magnesium sulfate, magnesia, magnesium borate, titanium diboride, carbon graphite, calcium sulfate, sapphire, silicon carbide and the like, preferably potassium titanate, Calcium carbonate and aluminum borate.

The whiskers used in the present invention can be used alone without surface treatment, but whiskers having a basic functional group on the whisker surface, preferably whiskers surface-treated with a surface treating agent, are used. By doing so, the transformation rate can be increased. Examples of the basic functional group at that time include a basic functional group formed by a primary or quaternary amine, a divalent metal complex, or the like, and preferably, an amino group is used.

The compound used for imparting a basic functional group to the whisker surface is not particularly limited as long as it can be covalently bonded to the whisker surface, but is preferably a silane coupling agent, more preferably a basic functional group. Silane coupling agent. Examples of the silane coupling agent include basic silane coupling agents such as 3- (2-aminoethoxylaminopropyl) -trimethoxysilane and 3-aminopropyl-triethoxysilane. It is not limited to these as long as it has a silane coupling agent.

The foreign gene used in the present invention is a nucleic acid used for transforming a plant, and may be DNA or RNA, but usually, DNA is used. Here, the term "transformation" refers to the introduction of a gene into a plant cell, regardless of whether or not the gene is expressed, but it is preferable that the gene be stably maintained in the plant cell. The gene may be a protein or peptide or an antisense RN.
It may encode A or the like and express these in plant cells, or may be inserted into a gene in a chromosome to inhibit the normal expression of that gene.

The form of the DNA used in the present invention may be any of double-stranded DNA and single-stranded DNA, circular DNA (plasmid), and linear DNA. When the gene of the present invention is introduced into a plant cell and then the gene is to be expressed, the gene or the promoter or terminator specific to the gene may be used as it is when it is operable, but it may be used efficiently in the plant cell. It may be replaced with a promoter or terminator that functions well or specifically.

For example, as a promoter functioning in plants, CaMV3 derived from cauliflower mosaic virus
55 promoter (Odell et al., 1988, "Pl
ant Mol. Biol. , Vol. 10, 263-
272), and the terminator includes a terminator derived from a cauliflower mosaic virus and a terminator derived from the nopaline synthase gene of Agrobacterium.

Examples of the foreign gene used in the present invention include, for example, herbicide resistance gene, insect resistance gene, virus resistance gene, disease resistance gene, storage protein gene,
Furthermore, a drug resistance gene and the like as a selection marker can be mentioned, but it is not particularly limited as long as it is a foreign gene that functions in a plant.

The exogenous gene used in the present invention can be used as it is, but the transformation rate can be increased by forming a complex that is electrostatically ion-bonded to a basic substance.

The basic substance capable of binding to a foreign gene to form a complex includes, for example, spermine,
Spermidine, di-lysine, tri-lysine, polylysine, di-arginine, tri-arginine, polyarginine, di-ornithine, tri-ornithine, polyornithine, histone H1, histone H2, histone H3, histone H4, and the like; The substance is not particularly limited as long as it has a positive charge in an aqueous solution, but polyornithine, spermine, spermidine, histone, di-lysine, and tri-lysine are preferred. These basic substances may be used as a mixture of two or more kinds.

These basic substances are used as foreign genes in order to keep the foreign genes stable in plant cells and to facilitate integration into chromosomes. As the amount of the basic substance with respect to the foreign gene, 0.1 to 300 μg, preferably 1 to 80 μg of the basic substance is used for 10 μg of the foreign gene.

Next, the liquid for dispersing plant cells or plant tissue, whiskers and foreign genes is used for the purpose of suspending plant cells and whiskers and dissolving the foreign genes. Distilled water, isotonic solution, Buffers, tissue culture media and the like can be mentioned.

As the isotonic solution at that time, for example, KCl,
A liquid obtained by adding an inorganic salt such as NaCl, CaCl ₂ , and MgCl ₂ to 0.01 to 7 M, preferably 0.5 to ₂ M is used. Examples of the buffer include phosphate buffer, Tris buffer, MES buffer and the like.

Examples of the medium for tissue culture include, for example, MS medium, MS medium, Gumborg's B5 medium, and R medium.
2 medium, white medium, niche-niche medium, N6 medium and the like.

When an isotonic solution or a medium for tissue culture is used, a saccharide is preferably added and used. Examples of the saccharide include sucrose, fructose, glucose, sorbitol, mannitol and the like. The concentration is 0.01 to 7
M, preferably 0.5-2M.

The pH of the liquid used in the present invention is from pH 4 to
8, preferably pH 5-7. Although the volume of the liquid used in the present invention is not particularly limited, the volume of the plant cell (compressed cell volume: Packed Cell Volume)
me, hereinafter abbreviated as “PCV”) is 10 μl to 2 ml, preferably 300 to 700 per 1 ml of PCV.
Use it after adjusting it to μl.

The volume of the plant cells used in the present invention is not particularly limited, but the amount of whiskers mixed in the liquid together with the plant cells is adjusted according to the volume of the plant cells.
Whisker 1 to 10 per 1 ml of PCV of plant cell amount
0 mg, preferably 4 to 40 mg.

The amount of the foreign gene or the complex of the foreign gene and the basic substance used in the present invention is 0.1 to 1000 μg, preferably 10 to 2 μg per 1 ml of PCV.
It is added so as to be 00 μg.

The plant cells, whiskers and exogenous gene thus obtained are added to the same container together with the liquid and mixed. As the container used in the present invention, for example, a centrifuge tube, a glass test tube, a polypropylene test tube, a petri dish, a flask, and the like can be used. However, the container is not limited thereto as long as it can be handled aseptically.

Plant cells, whiskers and foreign genes are:
The container is shaken and agitated so as to be uniformly mixed and dispersed in the liquid, but there is no need to vigorously agitate. In this way, a mixture of plant cells, whiskers, and foreign genes dispersed in a liquid (hereinafter, abbreviated as “mixture”) can be obtained.

[2] Step (b) Centrifugation This step is a step of attaching whiskers to plant cells by centrifuging the mixture. In the centrifugal treatment in the present invention, the centrifugal acceleration is 3,000 to 50,000 ×
g, preferably 10,000 to 30,000 × g,
The centrifugation time is 10 seconds to 40 minutes, preferably 5 to 10 minutes. Further, in order to increase the amount of whisker adhering to the plant cells, the same centrifugation treatment may be repeated 1 to 20 times, preferably 3 times.

[3] Step (c) Ultrasonic Treatment In this step, whiskers are vibrated by irradiating the mixture with ultrasonic waves after centrifugation, the cells are perforated by the whiskers, and the foreign gene is passed through the cells through the pores. This is the step of introducing it inside.

The ultrasonic treatment used in the present invention is carried out under milder conditions than those of the above-mentioned prior art, that is, when the frequency is 1 k to 1 k.
1 MHz, preferably 10 to 60 kHz, irradiation time 0.2 seconds to 20 minutes, preferably 30 seconds to 2 minutes, intensity is 0.01 to 10 W / cm ² , preferably
Irradiation is performed at 0.1 to 1 W / cm ² .

After sonication, the mixture is allowed to stand for a time sufficient to allow the foreign gene to enter and diffuse into the cells. The temperature at that time is 0 to 28 ° C, preferably 4 ° C,
The standing time is 1 minute to 3 hours, preferably 5 to 10 minutes.

According to the present invention, the whisker is vibrated in combination with the use of the whisker and the ultrasonic treatment, so that the whisker can penetrate into the cell under milder conditions than in the prior art. It is presumed that high transformation efficiency can be obtained without giving.

[4] Step (d) Plant Regeneration Step This step is a step of producing a transformed plant by regenerating a plant from the transformed cells obtained in the step (c).

When a foreign gene that can be expressed is introduced into a plant cell in performing this step, as described above, after centrifugation and sonication, the plant cell is cultured in advance to express the foreign gene. Let it.

The mixture subjected to the ultrasonic treatment as described above can be cultured as it is, but preferably, the mixture is washed to remove whiskers and foreign genes from the mixture. The washing solution used at this time includes distilled water, an isotonic solution, a buffer solution, a culture medium, and the like. Preferably, the above-mentioned isotonic solution or culture medium is used.

The washing may be carried out as long as the plant cells can be taken out of the mixture by an operation such as extinction. The operation is not particularly limited. For example, the mixture may be washed using a node such as a 40 μm nylon mesh. After washing, the plant cells can be removed.

The plant cells thus obtained are cultured using a plant tissue culture medium. The medium used at this time is, if necessary, the plant tissue culture medium described above,
As plant hormones, for example, auxins such as 2,4-PA, naphthalene acetic acid, indole acetic acid, and cytokinins such as benzyl adenine and kinetin are used alone or in combination. As a carbon source, for example, sucrose,
Glucose or the like can be added and used, but the medium can be selected according to the plant species to be transformed.

Cultivation is performed in a liquid medium or a solid medium.
5 to 30 ° C, preferably 20 to 28 ° C, and the culture period is 6
By performing the treatment for a period of time to 7 days, preferably for 2 to 3 days, it is possible to obtain a dividing cell in which a foreign gene is expressed in a plant cell.

At this time, these divided cells contain a mixture of transformed and non-transformed cells into which a foreign gene has been introduced. It is desirable to select cells from dividing cells and to separate them.

For example, by having a drug resistance gene serving as a selection marker, for example, an antibiotic hygromycin resistance gene, on the foreign gene used for introduction,
Transformed cells can be selected using the effect of inhibiting the metabolism of cells by the drug. To make this selection,
The dividing cells obtained above are placed or suspended in a solid or liquid selection medium consisting of a plant tissue culture medium to which a drug serving as an indicator has been added in advance, and are suspended for 20 to 60 days.
Culture is preferably performed for 30 to 40 days.

As the drug added to the selection medium, for example, hygromycin, kanamycin and the like can be used.
The concentration is 1 to 300 mg / liter, preferably 25 to 5
Although used at 0 mg / liter, the type and concentration of the drug can be selected according to the plant type, and are not particularly limited thereto.

The number of transformed cells varies depending on the type of foreign gene. For example, when the hygromycin resistance gene is used, the number of transformed cells tends to be smaller than that of the GUS gene.

The method for regenerating a plant in the present invention includes:
The transformation can be performed by placing the transformed cells obtained as described above in a known plant regeneration medium.

The transformed cells placed on the medium for plant regeneration are at 15 to 30 ° C., preferably 20 to 28 ° C., and irradiated with light at 500 to 2,000 lux, preferably 800 to 2,000 lux.
When the cells are cultured at 1,000 lux for a culture period of 20 to 60 days, preferably 30 to 40 days, a foreign gene of interest is introduced from individual cells, and transformed plants are regenerated.

[0055]

EXAMPLES The present invention will now be described specifically with reference to Examples, but the scope of the present invention is not limited to these Examples.

[0056]

Example 1 <1> Step (a) (1) Preparation of Test Plant Cells Rice (Rice variety: Harumoto) is hulled from mature rice seeds, placed in a 70% ethanol solution for 10 seconds, and then available chlorine (about 1%). After being immersed in a sodium hypochlorite solution for 6 minutes for sterilization, it was washed with sterilized water.

The inorganic component composition of the MS medium was determined to be 30 g / sucrose.
Liter, 2,4-PA 2mg / liter, agar 8g
Medium (pH 5.8) obtained by the addition of
A solid medium was prepared by solidifying in a 0 mm petri dish.

Nine seeds obtained above were placed on this solid medium in a petri dish and kept at 28 ° C. for 14 days in a light place (2,000
Lux, illumination for 16 hours per day) to obtain calli. 30 g / l of sucrose, 2 mg / l of 2,4-PA, 2 casamino acids
g / L of the liquid medium (pH 5.8) was added to a 100 ml Erlenmeyer flask, and 50 ml of the liquid medium was sterilized by an autoclave to prepare a liquid medium (hereinafter, this liquid medium was referred to as "R2D2 medium"). Abbreviated.)

The callus obtained above was cut out from the endosperm into this R2D2 medium, and 10 of the calli were transplanted per flask. At a temperature of 28 ° C., in a light place (2,000 lux, illumination for 16 hours per day), Rotary shaker (100
(rpm / min) to obtain suspension cultured cells. The suspension culture cells were added to PCV every 7 days.
Was transplanted to a fresh R2D2 medium and subcultured. After 28 days of subculture, the rice calli were sieved to 1 mm or less using a 1-mm stainless steel mesh sieve.
3 ml of PCV was obtained per petri dish.

The obtained rice callus of 1 mm or less is R2D
The plate was washed three times with 2 media and subjected to a test.

(2) Preparation of Whisker A potassium titanate whisker (product “LS20” manufactured by Titanium Industry Co., Ltd.) (5 mg) was placed in a 1.5 ml tube (manufactured by Eppendorf), and 0.5 ml of ethanol was added thereto. After standing overnight, ethanol was completely evaporated to obtain sterilized whiskers. Add 1 ml of sterilized water to the tube containing the whisker, mix well, and add
The mixture was centrifuged at 00 rpm / min for 5 minutes, the supernatant water was discarded, and the whiskers were washed. After performing this washing operation three times, 0.5 ml of R2D2 medium was added into the same tube to obtain a whisker suspension.

(3) Preparation of Foreign Gene As the foreign gene, pBI221 (manufactured by Clonetech) having the β-glucuronidase (GUS) gene was used.

The plasmid (PBI221) contained 1 mg / m
l of TE buffer (Tris-HCl 10 mM,
EDTA 1 mM, pH 8.0).

(4) GUS Gene Transfer Operation 250 μl of the callus of 1 mm or less obtained in (1) above was placed in a tube containing the whisker suspension obtained in (2) above with a PCV, followed by stirring. 1 at 1,000 rpm / min
Centrifugation was performed for 0 second to precipitate callus and whisker, and the supernatant was discarded to obtain a mixture of callus and whisker.

Further, 10 μl of R2D2 medium was added to 20 μl (containing 20 μg) of the GUS gene solution obtained in the above (3).
After adding l and mixing, the mixture was added to the tube containing the mixture of callus and whisker described above, and the mixture to which callus, whiskers and the exogenous gene were added was sufficiently shaken to obtain a mixture.

<2> Step (b) Next, a tube containing this mixture was placed at 18,000 × g
For 5 minutes, followed by centrifugation and shaking again. The operation of centrifuging and shaking again was performed three times.

<3> Step (c) (1) Ultrasonic treatment The tube containing the mixture thus obtained is sufficiently immersed in a bath of an ultrasonic generator (tub type: using water as a medium). And irradiate with ultrasonic waves at a frequency of 40 kHz and an intensity of 0.25 W / cm ² for 1 minute.
It was left at 4 ° C. for 30 minutes.

The mixture was subjected to the ultrasonic treatment in this manner to make the mixture 40 μm.
The mixture was filtered through a nylon mesh having pores, and further washed with 5 ml of R2D2 medium. This operation was repeated three times.
Calli into which the S gene was introduced were obtained.

(2) Culture of dividing cells The callus into which the GUS gene was introduced was placed in a 3.5 cm petri dish, 3 ml of R2D2 medium was added, and the mixture was placed at 28 ° C. in a light place (2,000 lux, illumination for 16 hours per day). hand,
The cells were cultured using a rotary shaker (50 rpm / min) to obtain dividing cells.

(3) Selection of Number of Transformed Cells The number of cells into which the GUS gene had been introduced was measured as follows. That is, in the culture of the dividing cells in the above (2), only the R2D2 liquid medium was removed on the second day of culture, only the callus was left in the petri dish, and 1 mM X-Gl
u (5-Bromo-4-chloro-3-indo
lyl-β-D-gluconide) solution 300μ
1 was added to a Petri dish, and left at 36 ° C. in a dark place for 24 hours to carry out an enzyme reaction. With this GUS assay,
Only the transformed cells in which the GUS gene was introduced and transformed in the calli were colored blue. These cells were selected under a microscope and the number of transformed cells was examined. The results are shown in Table 1.

[0071]

Example 2 1 g of potassium titanate whisker (“LS20” manufactured by Titanium Industry Co., Ltd.) was placed in a 500 ml eggplant-shaped flask, 100 ml of toluene was added, and 3- (2-aminoethoxylaminopropyl) -trimethoxy was further added. After 1 g of silane (coupling agent) was added and dissolved, the temperature of the toluene in the flask was adjusted to 120 ° C., and the mixture was stirred in a silicon oil bath to distill off the toluene to obtain a slurry.

After the reaction, the slurry was washed with 90% methanol to remove excess coupling agent. Further, the residual methanol used for washing was completely distilled off with a rotary evaporator to obtain a surface basic whisker.

The surface basic whisker obtained above was used in Example 1.
After sterilization in the same manner as described above, an R2D2 medium was added to obtain a mixture.

TE buffer 2 containing 20 μg of GUS gene
To 0 μl, 40 μl of R2D2 medium was added, and 2.5 μg of polyornithine, 50 μg of spermidine, 18 μg of spermine, and 20 μg of histone H2 were individually dissolved in 60 μl of R2D2 medium and added to this solution at room temperature.
After standing for 4 minutes, a basic complex of four types of GUS genes was prepared.

Using a mixture of these basic complex of GUS gene and the above-mentioned surface basic whisker, transformed cells were obtained in the same manner as in Example 1, and the number of transformed cells was determined by GUS assay. . The results are shown in Table 1.

[0076]

[Comparative Example 1] G of divided cells was transferred in the same manner as in Example 1, except that only centrifugation was performed at the time of gene transfer, and sonication was omitted.
A US assay was performed to obtain transformed cells. The results are shown in Table 1.

[0077]

Comparative Example 2 Transformed cells were obtained by GUS assay of dividing cells according to Example 1, except that the centrifugal treatment was omitted during gene transfer and only ultrasonic treatment was performed. The results are shown in Table 1.

[0078]

[Comparative Example 3] The addition of whiskers was omitted during gene transfer,
Callus PCV 2 of 1 mm or less in 1.5 ml tube
50 μl, foreign gene solution 20 μl, R2D2 medium 10
A GUS assay of dividing cells was performed in the same manner as in Example 1 except that a mixture obtained by adding 0 μl was used to obtain transformed cells. The results are shown in Table 1.

[0079]

[Table 1]

[0080]

Example 3 (1) Preparation of Test Plant Cells ”was performed according to Example 1. (2) Preparation of whisker was performed according to Example 1. (3) Preparation of Foreign Gene As the foreign gene, a plasmid pCH carrying a hygromycin resistance gene (available from the University of Tokyo) was used. (4) The operation of introducing the hygromycin resistance gene was performed according to Example 1.

(5) Culture of dividing cells Hygromycin-resistant callus was placed in a 3.5 cm petri dish, 3 ml of R2D2 medium was added, and the mixture was incubated at 28 ° C. in a light place (2,000 lux, illumination for 16 hours per day).
The cells were cultured using a rotary shaker (50 rpm / min) to obtain dividing cells.

(6) Selection of transformed cells Culture was carried out according to the above (5). On the third day of the culture, 3 ml of a suspension of these divided cells was treated with 2 mg / l of 2,4-PA and 30 g of sucrose. 30 ml of N6 medium (pH 5.8) containing 5 g / l, gellite 3 g / l, and hygromycin 50 mg / l were spread evenly on a solid medium solidified in a 9 cm-diameter dish, and then suspended with a pipette. The turbid liquid was sucked out.

This was heated at 28 ° C. in a light place (2,000 lux,
(Lighting for 16 hours per day) for 20 days to obtain hygromycin-resistant transformed cells.

(7) Regeneration of Plant from Transformed Cells 169 transformed cultured cells (about 5 m in diameter) obtained as described above
m), pendyl adenine 2 mg / l, naphthalene acetic acid 1 mg / l, sucrose 30 g / l, sorbitol 30 g / l, gellite 3 g / l
30 ml of MS medium containing 10 liters was placed on a regeneration medium solidified in a 9 cm diameter petri dish, and 10 plates were placed per petri dish.
After culturing for 30 days (lighting for 16 hours per day), shoots and roots were regenerated from the transformed cells.

Regenerated shoots and shoots containing roots (length 10 to 3)
(Grown to 0 mm) in 3% sucrose and 50 ml of MS medium containing 3 g / l of gellite in a plant box (length 5c).
mx 5 cm x 10 cm height), each of which was transplanted to a solid medium solidified and cultured for 20 days to obtain 135 transformed plants. Table 2 shows the results.

(8) Genetic analysis of transgenic plants PCR (polymerase chain react)
The plant carrying the hygromycin resistance gene was analyzed by the Tion method.

50 mg of leaves of the regenerated plant obtained in the above (7)
Into a 1.5 ml microtube and 10 mM
20 mM Tris-HCl buffer containing EDTA (p
H7.5) 300 μl was added, and this was ground,
20 ml of 0% SDS was added, and the mixture was heated at 65 ° C. for 10 minutes. 100 μl of 5M potassium acetate was added thereto, and the mixture was placed on ice for 20 minutes, and then centrifuged at 1,7000 × g for 2 minutes.
After centrifugation for 0 minutes, 200 μl of isopropanol was added to the obtained supernatant, and the mixture was stirred by inversion.
The mixture was centrifuged at a centrifugal acceleration of 00 × g for 20 minutes, and the precipitate was dried under reduced pressure and dissolved in 100 μl of TE buffer to obtain DNA.

Oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 1 and 2 were synthesized as primers for PCR based on the sequence of the known hygromycin resistance gene. Next, 1 μM of each of these oligonucleotides was used as a primer, and 5 μl of the plant-derived DNA was used as a template to prepare a reaction solution containing these (10 mM Tris).
-HCl (pH 8.3), 1.0 mM MgCl ₂ , 5
0 mM KCl, 0.01% gelatin, pH 8.3, d
NTP 0.2 mM each, Taq DN
A polymerase 2.5 units) 100μ
At l, an amplification reaction was performed. The reaction solution is a PCR kit (P
It was prepared using CR Amplification Kit (manufactured by Takara Shuzo Co., Ltd.).

The amplification reaction was performed using a PCR reaction device (Progr
am Temp Control System PC-
700 (manufactured by Aztec)) at 94 ° C.
Minutes, annealing 60 ° C, 30 seconds, extension (extent
ion) The reaction conditions of 72 ° C. and 1 minute were repeated 30 times.

When the PCR reaction solution was analyzed by agarose electrophoresis according to a conventional method, an amplified DNA band of 375 bp was confirmed from DNA extracted from the regenerated plant.
From this DNA band pattern, the rice regenerated plant 135
It was confirmed that the hygromycin resistance gene was integrated into all of the individuals. Table 2 shows the results.

[0091]

Example 4 (1) Preparation of Test Plant Cells Immature embryos (1 to 1.2 mm long) on the 10th day of mating of corn (variety A188) were taken out under aseptic conditions, and sucrose was added to the inorganic component composition of the N6 medium. 20g / liter, 2,4-P
A A solid obtained by adding a medium (pH 5.8) obtained by adding 2 mg / L, L-proline 2.9 g / L, casamino acid 100 mg / L, and agar 9 g / L in a petri dish having a diameter of 9 cm. Medium (hereinafter referred to as "N
9S per petri dish was placed on a 6S2 medium) and cultured at 28 ° C. in a dark place for 21 days to obtain calli.

"(2) Preparation of whisker" and "(4)
The procedure for introducing the hygromycin resistance gene is the same as in Example 1, and the procedure of "(3) Preparation of foreign gene" is the procedure in Example 2.
It went according to.

(5) Culture of dividing cells Hygromycin-resistant calli were individually tipped with N6
The cells were placed on an S2 medium and cultured in the dark at 28 ° C. for 7 days to obtain dividing cells.

(6) Selection of Transformed Cells Cultured according to the above (5), the calli on day 7 of the culture were directly transferred to an N6S2 medium supplemented with 50 mg / liter of hygromycin without dividing, and the culture was incubated at 28 ° C. in the dark. After culturing for 30 days, transformed cells resistant to hygromycin were obtained.

(7) Regeneration of Plants from Transformed Cells 101 transformed cultured cells (grown to a diameter of about 5 mm) obtained by the above (6) were prepared using 10 g / l of sucrose and 3 g / l of gellite. 6 callus pieces per 9 cm diameter petri dish were placed on a N6 medium (pH 5.8) having a concentration of 1/2 inorganic salt, and placed at 28 ° C. in a light place (2,000 lux, illumination for 16 hours per day) for 30 days. After culturing for a day,
Buds and roots regenerated from the transformed cultured cells.

Regenerated buds and buds containing roots (length 10 to 3)
(Grown to 0 mm) was transplanted one by one to a solid medium solidified in a plant box (5 cm × 5 cm × 10 cm in height) containing 50 ml of an MS medium containing 30 g / l of sucrose and 3 g / l of gellite. After culturing for 30 days, 30 transformed plants were obtained. Table 2 shows the results.

(8) Genetic Analysis of Transformed Plant Analysis was carried out according to the method of Example 2, and it was confirmed that the hygromycin resistance gene was incorporated into 26 out of 30 corn regenerated plants. . Table 2 shows the results.

[0098]

Comparative Example 4 The number of hygromycin-resistant transformed cells in dividing cells was investigated in the same manner as in Example 3 except that only centrifugation was performed at the time of gene transfer and sonication was omitted. It was shown to.

[0099]

Comparative Example 5 The number of hygromycin-resistant transformed cells in dividing cells was examined in the same manner as in Example 3 except that centrifugation was omitted and only ultrasonic treatment was performed during gene transfer. It was shown to.

[0100]

[Comparative Example 6] The addition of whiskers was omitted during gene transfer,
Callus PCV 2 of 1 mm or less in 1.5 ml tube
50 μl, foreign gene solution 20 μl, R2D2 medium 10
Except that a mixture obtained by adding 0 μl was used, the number of transformed hygromycin-resistant transformed cells was examined in the same manner as in Example 3, and the results are shown in Table 2.

[0101]

Comparative Example 7 The number of transformed hygromycin-resistant transformed cells was examined in the same manner as in Example 4 except that only centrifugation was performed at the time of gene transfer and sonication was omitted. It was shown to.

[0102]

Comparative Example 8 The number of hygromycin-resistant transformed cells in dividing cells was examined in the same manner as in Example 4 except that the centrifugal treatment was omitted and only the ultrasonic treatment was performed at the time of gene transfer. Table 2 shows the results. It was shown to.

[0103]

Comparative Example 9 The addition of whiskers was omitted during gene transfer,
Callus PCV 2 of 1 mm or less in 1.5 ml tube
50 μl, foreign gene solution 20 μl, N6S2 medium 10
Except that a mixture obtained by adding 0 μl was used, the number of transformed hygromycin-resistant transformed cells was examined in the same manner as in Example 4, and the results are shown in Table 2.

[0104]

Example 5 (1) Preparation of Test Plant Cells 70 mature seeds of turfgrass bentgrass (variety: Pencloth)
% Ethanol solution for 10 seconds, and then immersed in about 1% available chlorine sodium hypochlorite solution for 20 minutes to sterilize, wash with sterile water, and add 30 g / l sucrose to the inorganic component composition of the B5 medium. A medium obtained by adding 3 mg / L of 2,4-PA and 3 g / L of gellite (p
H5.8) was solidified in a petri dish having a diameter of 9 cm, and solidified on a solid medium (hereinafter, abbreviated as “B5D3 medium”).
100 petri dishes are placed per petri dish, 28 ° C, light place (2,
000 lux, illumination for 16 hours per day) for 60 days to obtain calli.

“(2) Whisker adjustment” and “(4)
The procedure for introducing the hygromycin resistance gene is the same as in Example 1, and the procedure of "(3) Preparation of foreign gene" is the procedure in Example 2.
It went according to.

(5) Culture of dividing cells Hygromycin-resistant calli were individually isolated with tweezers using B5
The cells were placed on a D3 medium and cultured at 28 ° C. in a light place (2,000 lux, illumination for 16 hours per day) for 7 days to obtain dividing cells.

(6) Selection of Transformed Cells Cultured according to the above (5), the calli on day 7 of the culture were transplanted directly to a B5D3 medium supplemented with 50 mg / liter of hygromycin without division, and the cells were cultured at 28 ° C. Light place (2,0
The cells were cultured for 40 days at 00 lux and illumination for 16 hours per day to obtain hygromycin-resistant transformed cells.

(7) Regeneration of Plants from Transformed Cells 57 transformed cultured cells (grown to a diameter of about 1 cm) obtained in the above (6) were obtained by sucrose 30 g / l, sorbitol 30 g / l, benzyl adenine 0 6 calli per 9 cm diameter petri dish were placed on an MS medium (pH 5.8) having a concentration of 1/2 inorganic salt containing 0.3 mg / l, naphthalene acetic acid 0.3 mg / l, and gellite 3 g / l. When cultivated at 28 ° C in a light place (2,000 lux, illumination for 16 hours per day) for 50 days,
Buds regenerated from the transformed culture cells.

The regenerated buds (grown to a length of 3 to 5 mm) were placed in a test tube (vertical 1) containing 30 g / L of sucrose and 3 g / L of gellite, and 30 ml of an MS medium having a 1/2 inorganic salt concentration.
5 cm x 4 cm in diameter)
The plants were transplanted one by one and cultured for 30 days to obtain 49 transformed plants having roots formed at the bases of the grown shoots. Table 2 shows the results.

(8) Genetic Analysis of Transformed Plant Analysis was carried out according to the method of Example 3, and it was confirmed that the hygromycin resistance gene was incorporated into 44 of 49 regenerated turfgrass plants. Was. Table 2 shows the results.

[0111]

[Comparative Example 10] The number of transformed hygromycin-resistant transformed cells was examined in the same manner as in Example 5, except that only centrifugation was performed at the time of gene transfer, and sonication was omitted.
The results are shown in Table 2.

[0112]

[Comparative Example 11] The number of transformed hygromycin-resistant transformed cells was examined in the same manner as in Example 5, except that the centrifugal treatment was omitted during the gene transfer and only the ultrasonic treatment was performed.
The results are shown in Table 2.

[0113]

Comparative Example 12 The addition of whiskers was omitted during gene transfer,
Callus PCV25 of 1mm or less in 1.5ml tube
0 μl, foreign gene solution 20 μl, B5D3 solution 100
The number of transformed hygromycin-resistant transformed cells was examined in the same manner as in Example 5, except that the mixture obtained by adding μl was used. The results are shown in Table 2.

[0114]

[Table 2]

[0115]

Industrial Applicability According to the present invention, a plant can be transformed easily and in a short time regardless of the species, variety and line of the plant, and a transformed plant can be efficiently produced.

[0116]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 26 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: NO sequence GCTGGGGCGT CGGTTTCCAC TATCCG 26

SEQ ID NO: 2 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Antisense: YES sequence CGCATAACAG CGGTCATTGA CTGGAGC 27

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) JICST file (JOIS) BIOSIS / WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A method for transforming a plant, comprising the following steps (a) to (c): (A) dispersing a plant cell or a tissue containing a plant cell in a liquid together with a foreign gene and whisker;
After the step (a), a centrifugal treatment is performed to attach whiskers to the plant cells. (C) After the step (b), an ultrasonic treatment is performed to pierce the plant cells with the whiskers. A step of introducing a foreign gene into the cell. 2. The method according to claim 1, wherein the foreign gene is a gene forming a complex with a basic substance. 3. A method for producing a transformed plant, comprising the following steps (a) to (d). (A) dispersing a plant cell or a tissue containing a plant cell in a liquid together with a foreign gene and whisker;
After the step (a), a centrifugal treatment is performed to attach whiskers to the plant cells. (C) After the step (b), an ultrasonic treatment is performed to pierce the plant cells with the whiskers. A step of introducing a foreign gene into the cell. (D) a step of regenerating a plant from a plant cell into which the foreign gene has been introduced in step (c) or a plant tissue containing the same.