JPH09252674A - Gene introduction method by microbe of genus agrobacterium and production method for transformed plant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently introduce a gene to the plant of the genus Brassica and to produce transformed plants by pre-culturing the tissue piece of the plant belonging to the genus Brassica, then immersing it in infection liquid containing the microbes of the genus Agrobaterium and culturing it under specified conditions. SOLUTION: After the tissue piece of the plant belonging to the genus Brassica such as Brassica rapa is pre-cultured in a tissue culture medium whose pH is 5.0-6.9, it is immersed in the infection liquid containing the microbe of the genus Agrobacterium, the immersed tissue piece is coexistingly cultured in the tissue culture medium and the gene is introduced to the plant of the genus Brassica. Also, it is preferable to provide a feeder cell in the tissue culture medium and it is preferable to set the values of X, Y and Z so as to satisfy the formula 82<X/10+(Y-19)<2> +Z<145 [X is the time (minute) to be immersed in the infection liquid; Y is a temperature ( deg.C) at the time of coexisting culture; Z is the period (hour) of the coexisting culture] for producing the transformed plant of the genus Brassica.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently introducing a gene into a plant of the genus Brassica using a microorganism of the genus Agrobacterium to produce a transformed plant.

[0002]

2. Description of the Related Art With the rapid progress of biotechnology, varieties having various characteristics have been bred by utilizing biotechnology technologies such as tissue culture, anther culture and cell culture. Furthermore, due to the development of recombinant DNA technology, varieties having characteristics that could not be expected in the past have been bred by introducing genes of other species that cannot normally be crossed.

In the United States, tomatoes (flavor savers) into which the antisense of polygalacturonase gene has been introduced are already on the market as tomatoes having a long shelf life. In addition, BT-Toxin-introduced insect-resistant potatoes, corn, herbicide-tolerant gene-introduced cotton, soybeans, and pumpkins with viral coat proteins were all approved as safe as food and put into practical use. Is in the stage of.

In Canada, herbicide-resistant rapeseed has finished safety evaluation and is in the stage of practical application. Commercialization of crops using these recombinant DNA technologies tends to progress more and more in the future. The production of varieties using recombinant DNA requires isolation of excellent genes and gene transfer technology into plants. Examples of methods for introducing genes into plants include the Agrobacterium method, electroporation method, and particle gun method. The Agrobacterium method can produce transformed plants at a high rate in Solanaceae and Cucurbitaceae plants, but has a low success rate in Brassicaceae plants, and the development of efficient gene transfer technology and regeneration technology is urgently needed. ing. Especially rape, Chinese cabbage,
Brassica rap, including turnips and tsenna
Since a) has a low ability to redifferentiate from tissue pieces, there are few reports that cells into which genes have been introduced could be differentiated to the individual level (Radke et al, Plant CellRep (1992) 11: 499-50.
5, Mukhopadhyay et al, Plant Cell Rep (1992) 11: 5
06-513, Jun et al. Plant Cell Rep. (1995) 14: 620-6
twenty five)).

[0005]

An object of the present invention is to provide an efficient method for producing a transgenic plant of the genus Brassica by a microorganism of the genus Agrobacterium, and to provide a recombinant DN.
Provided is a method for producing a variety of Brassica belonging to the genus Brassica.

[0006]

As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that p
It was found that the gene transfer rate was remarkably improved by setting H to a specific value or by adding a feeder cell to the tissue piece culture medium. Further, the immersion time in the infection solution, the temperature at the time of coculture, the coculture It was found that the three parameters of the period are closely related, and that the production efficiency of the transformed plant is remarkably improved by making these three specific values, and the present invention was completed based on these findings.

That is, according to the present invention, a tissue piece of a Brassica plant is pre-cultured in a tissue culture medium and then immersed in an infection solution containing a microorganism of the genus Agrobacterium. A method for introducing a gene into a plant of the genus Brassica by co-culturing in a culture medium, wherein the pH of the tissue piece culture medium is set to 5.0 to 6.9 Is.

In the present invention, a tissue piece of a Brassica plant is pre-cultured in a tissue culture medium, and then immersed in an infection solution containing a microorganism of the genus Agrobacterium. A method for introducing a gene into a plant of the genus Brassica by co-culturing in a culture medium, which is characterized in that the tissue piece culture medium contains feeder cells.

Further, according to the present invention, the tissue piece of the plant of the genus Brassica is pre-cultured in a tissue piece culture medium and then immersed in an infection solution containing a microorganism of the genus Agrobacterium, and the tissue piece after the immersion is the tissue piece. In a method of producing a transformed plant of the genus Brassica by co-culturing in a culture medium, then inducing callus from the tissue piece after co-culturing and re-differentiating the obtained callus, the following formula 82 <x / 10 + (y-19) ² + z <145 [where x is the time (minutes) for immersion in the infection solution, y is the temperature (° C.) during co-culturing, and z is the period (hours) during co-culturing]] The method for producing a transgenic plant of the genus Brassica is characterized in that x, y, and z are set to

Hereinafter, the present invention will be described in detail. The gene transfer method of the present invention, after pre-culturing a tissue piece of a Brassica plant in a tissue piece culture medium, it is immersed in an infection solution containing a microorganism of the genus Agrobacterium, and the tissue piece after immersion is the tissue piece culture. The gene is introduced into a Brassica plant by co-culturing in a medium. Further, the method for producing a transformed plant of the present invention produces a transformed plant of the genus Brassica by inducing callus from a tissue piece into which a gene has been introduced by the above method and redifferentiating the obtained callus.

The plant of the genus Brassica used herein may be any species as long as it belongs to the genus Brassica, and for example, brassica rapa, brassica oleasia and the like can be used. Among these species, brassica / rapa can be exemplified as a preferable species. Among the plants belonging to Brassica rapa, Chinese cabbage (B. rapa ssp. Pekinensis) and turnip (B.
rapa ssp. rapa), tsukena (B.rapa ssp. chinensis)
) Can be illustrated as a particularly preferable plant. Preferred varieties include friendly vegetables (Kaneko Co., Ltd.), Beni moss (Sakata seeds Co., Ltd.), Osome (Takii Seed Co., Ltd.) for Tsukena, and Honko Daimaru Kabu (Watanabe Seed Farm ( ), Japanese vegetable turnip (Takii Seed Co., Ltd.), Seigoin turnip (Takii Seed Co., Ltd.), Chinese cabbage, Strong C
R (Watanabe Seed Co., Ltd.), Kofu (Sakata Seed Co., Ltd.)
Etc. can be exemplified.

The tissue piece of the plant used for the pre-culture may be any one as long as it has the ability to differentiate to the individual level after gene introduction, and examples thereof include flower stems, leaves, cotyledon pieces, hypocotyls and the like. Can be used.

The tissue piece culture medium may be any medium as long as it contains components necessary for culturing plant tissue pieces, for example, carbon sources, nitrogen sources, inorganic salts, solid agents, plant hormones and the like. As the carbon source, for example, sucrose, glucose or the like can be used, and as the inorganic salts, MS
Inorganic salts, B5 inorganic salts, etc. can be used, agar, gellite, etc. can be used as solid agents, and 2,4-D, α-naphthalene acetic acid (N
AA), benzyladenine (BA), zeatin (Zeati
n) or the like can be used. Further, the tissue piece culture medium preferably contains a feeder cell. The feeder cell improves the gene transfer rate of Brassica plants. As the feeder cell, for example, a suspension of tobacco or tomato can be used. The concentration of feeder cells in the tissue culture medium was 1.5 ml / 90 cm Petri dishes.
It is preferable to set the degree. The pH of the tissue culture medium is
It is preferably 5.0 to 6.9, more preferably 5.2 to 5.8. By setting the pH of the medium in such a range, the gene transfer rate of Brassica plants can be improved. The temperature during the pre-culture is not particularly limited as long as it does not adversely affect the tissue piece of the plant, but is 20 to 28 ° C.
It is preferred that

The microorganism of the genus Agrobacterium used is
Although not particularly limited, Agrobacterium tumefaciens is preferable, and CIB542 / A136 strain or EHA101 strain is particularly preferable. The Agrobacterium EHA101 strain has the binary vector pIG12Hm. The T-DNA region of this vector is shown in FIG. As shown in FIG. 1, this vector has a GUS gene as a reporter gene, a kanamycin resistance gene (KmR) and a hygromycin resistance gene (HygR) as selection marker genes. The concentration of the microorganism of the genus Agrobacterium in the infectious solution is not particularly limited, but is preferably about 1.2 × 10 ⁸ cells / ml.

The time of immersing the plant tissue piece in the infectious solution, the temperature at the time of co-cultivation, and the period of co-cultivation are factors that greatly affect the gene transfer rate and the subsequent redifferentiation rate. From the viewpoint of improving the gene transfer rate, it is preferable to lengthen the immersion time and the co-culture period and increase the temperature during the co-culture. However, by increasing the immersion time and the coculture period and increasing the temperature during the coculture, the redifferentiation rate of the tissue piece into which the gene has been introduced is reduced. Therefore, in order to improve the production efficiency of transformed plants,
It is necessary to determine the suitable range by comprehensively considering the immersion time, the culture temperature, and the culture period. Specifically, the immersion time is x
Minutes, the culture temperature is y ° C., and the culture period is z hours,
It is preferable to set x, y, and z so as to satisfy the following expression 82 <x / 10 + (y-19) ² + z <145, so that 98 ≤ x / 10 + (y-19) ² + z ≤ 113 is satisfied. It is more preferable to set. Immersion time,
The culture temperature and the culture period may take any values as long as the above formula is satisfied, but the immersion time is preferably 10 to 30 minutes, and the culture temperature is preferably 20 to 30 ° C.
The culture period is preferably 24 to 72 hours.

The tissue piece into which the gene has been introduced, after inducing callus, is finally differentiated to the individual level via adventitious buds, shoots and the like. Such regeneration can be performed according to a conventional method.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION

[0018]

EXAMPLES First, the composition of the medium used in Examples and Test Examples is shown below.

<Seeding medium> MS inorganic salt, pyrimidine hydrochloride: 50 μg / L, nicotinic acid: 50 μg / L, glycine:
200 μg / L, phytoagar: 0.6%, pH 5.8 <Tissue piece culture medium> MS inorganic salt, myo-inositol: 1
00 mg / L, thiamine hydrochloride: 1.3 mg / L, KH ₂ PO ₄ :
200 mg / L, 2,4-D: 1 mg / L, sucrose: 3%,
Phyto agar: 0.6%

<Callus induction medium> B5 inorganic salt, B5 vitamin, 2,4-D: 1 mg / L, sucrose: 3%, phytoagar: 0.6%, pH 5.8 <adventitious bud formation medium> B5 inorganic salt, B5 vitamins, BA:
3 mg / L, zeatin: 1 mg / L, sucrose: 1%, phytoagar: 0.6%, pH 5.8 <Adventitious bud maturation medium> B5 inorganic salt, B5 vitamin, sucrose: 1%, phytoagar: 0.6% pH 5.8

<Rooting medium> B5 inorganic salt, B5 vitamin,
Sucrose: 1%, Phyto agar: 0.6%, IBA:
2 mg / L, pH 5.8 <YBE medium> Bactopeptone: 5 g / L, yeast extract: 1 g / L, beef extract: 5 g / L,
MgCl ₂ : 0.5 g / L, sucrose: 5 g / L, bacto agar: 1.5%

Test Example 1 The gene transfer rate of a microorganism belonging to the genus Agrobacterium largely depends on the Vir gene expression of this microorganism. It is known that the expression of Vir gene is further enhanced by low pH, addition of monosaccharides, addition of phenol compounds such as acetosyringone. Therefore, changing the pH of the tissue culture medium,
Sugar, acetosyringone, or feeder cells were added to the medium to examine the effect on the gene transfer rate.

First, the pH of the tissue culture medium was adjusted to 5.8 or 5.2, and glucose, acetone syringone, or feeder cells were added to the medium. Glucose was added at a concentration of 0.1 M in the medium, and acetosyringone was added at a concentration of 100 μM in the medium. Also,
Tobacco (BY-2) was used as the feeder cell so that the concentration of the feeder cell in the medium was 1.5 ml / 90 cm Petri dish.

Next, 6 after sowing the Komatsuna variety (Osome)
5-10 mm hypocotyls were collected from the seedlings on the -7th day, placed on the tissue piece culture medium whose pH and components were adjusted as described above, and precultured for 1 day. The hypocotyl after preculture was immersed in an infection solution containing Agrobacterium EHA101 (pIG121Hm) for 10 minutes. This infectious solution was obtained by culturing Agrobacterium EHA101 (pIG121Hm) overnight in a YBE liquid medium, and diluting the culture solution having a turbidity of OD ₆₈₀ = 1.0 tenfold with a tissue culture medium. Was prepared by.
The hypocotyl soaked in the infection solution is returned to the precultured medium,
Co-culture was carried out at 0 ° C for 2 days.

Since the binary vector pIG121Hm has a GUS gene, when gene transfer occurs, the hypocotyl of the hypocotyl becomes positive for GUS activity. Therefore, it was judged whether or not gene transfer occurred using GUS activity as an index. The results are shown in Table 1.

[0026]

[Table 1]

As shown in Table 1, the gene transfer rate was relatively higher when the medium pH was 5.2 than 5.8. The gene transfer rate was also improved by adding feeder cells to the medium. The addition of sugar to the medium did not affect the gene transfer rate.

[Test Example 2] The effects on the gene transfer rate were examined by changing the temperature and period of co-culture and the time of immersion of the hypocotyl in the infectious solution. The same hypocotyl as in Test Example 1 was added to a feeder cell (tobacco [BY-2]) and placed in a tissue piece culture medium adjusted to pH 5.2, and precultured for 1 day, then Test Example 1 The hypocotyl after preculture was immersed in the infection solution prepared in the same manner as in 10 minutes or 30 minutes. After the immersion, the hypocotyl was returned to the tissue piece culture medium and cocultured for 1 to 3 days. The temperature during co-cultivation was 25 ° C or 28 ° C. The GUS activity after co-culture was examined in the same manner as in Test Example 1. Table 2 shows the results.

[0029]

[Table 2]

The gene transfer rate of Agrobacterium tended to be higher at the coculture temperature of 28 ° C than at 25 ° C.
In addition, the gene transfer rate improved as the co-culture period was extended from 1 day to 3 days. Immersion time in infection solution is 30
The gene transfer rate was further improved by setting the time to minutes.
Generally, the culture of microorganisms of the genus Agrobacterium is 28 ° C,
It takes 24 hours. 28 ° C appears to be the optimum temperature for Agrobacterium growth. It is considered that the gene transfer rate tends to increase in proportion to the growth rate of Agrobacterium.

[Test Example 3] As shown in Test Example 2, the infection rate can be increased to 74.5% by soaking the infection solution for 30 minutes and co-culturing at 28 ° C. for 3 days. It was However, due to the growth of Agrobacterium microorganisms in co-culture, the redifferentiation rate from the hypocotyl may decrease, and the final transformation efficiency may decrease. Therefore, the production rate of transformed plants under the six infection conditions that showed a high gene transfer rate was examined. Table 3 shows the results
Shown in

[0032]

[Table 3]

When the co-cultivation temperature was 25 ° C., the growth of microorganisms of the genus Agrobacterium did not affect the regeneration from the hypocotyl. As the gene transfer rate increases, the number of kanamycin-resistant callus and shoots obtained increases,
When the immersion time in the infection solution was 30 minutes and the coculture period was 3 days, a transformant production rate of 5% was obtained.

On the other hand, when the co-cultivation temperature was 28 ° C., the growth of microorganisms of the genus Agrobacterium greatly affected the regeneration from the hypocotyl. With the increase in infection rate,
The number of hypocotyls that die of browning increased rapidly, and the number of kanamycin-resistant callus and shoots obtained tended to decrease.

As described above, when the co-culture temperature is increased, the gene transfer rate is improved, but the redifferentiation rate is lowered, so that the final production rate is not so good. On the contrary, when the co-culture temperature is lowered, the redifferentiation rate is improved, but the gene transfer rate is lowered and the production rate is not so good. Therefore, in addition to the co-culturing temperature, the production rate is set by setting the sum of the three parameters that affect the gene transfer rate and the redifferentiation rate, that is, the immersion time in the infection solution and the co-cultivation period, within a certain range. It is thought that it can be improved. However, the immersion time has the least effect on the gene transfer rate and the redifferentiation rate, so it is divided by 10. Also, since the rate of change in the culture temperature is small compared to other parameters, the growth of Agrobacterium microorganisms decreases. In order to increase the temperature from 19 ° C., the value is subtracted at 19, and the value is further squared, and the following equation is set.

Immersion time [min] / 10 + (incubation temperature [° C])
-19) ² + Culture period (hours) As shown in Table 3, when the value of this formula is larger than 82 and smaller than 145, the production efficiency of transformed plants is high.

[Example 1] Seeds of one cultivar "Osome" (Takii Seed Co., Ltd.) of B. rape ssp. Chinensis were dipped in 70% ethanol for 2 minutes, and then 1 drop of Tween 20 was added 1 % Sodium hypochlorite solution for 15-20 minutes and washed 3 times with sterile water. The seeds that had been sterilized were sowed in 15 seeds each in an agripot containing a seeding medium, and then subjected to 6-7 at 25 ° C., 45-55 μE, and 16 hours photoperiod.
Nurtured for days.

From the seedlings grown to 4-5 cm 6-7 days after seeding, 5-10 mm hypocotyls were cut out and arranged in a tissue piece culture medium (50 hypocotyls / dishes). To the tissue piece culture medium, 1.5 mL of a suspension culture solution of tobacco (BY-2) subcultured 4 days ago was dispensed in advance, and a sterile filter paper was laid on it. . Petri dish (9 cm) is a surgical tape (3M
Sealed with Micropore) and precultured at 25 ° C. in the dark for 24 hours.

Agrobacterium EHA101 (pIG121Hm) Transferred from glycerol stock to YBE medium, 28 ° C., 3
Incubated for days. Agrobacterium EHA101
YBE liquid medium containing Kanamycin and Hygromycin (Km50mg / L, Hyg50mg / L)
28, until the turbidity reaches OD ₆₈₀ = 1.0.
Culturing was carried out with shaking at 200 rpm for 18 to 24 hours. 1.5 mL of the above-mentioned microbial suspension was added to a 9 cm dish in which 13.5 mL of the tissue piece culture medium whose pH was adjusted to 5.2 was dispensed,
An infection solution was created. Hypocotyls were immersed in the infection solution (200 / dishes) and shaken at room temperature and 40 rpm for 30 minutes. After sucking the infection solution with a Pipetman, the hypocotyl was transferred to a sterile Kim towel to remove excess infection solution. Return the hypocotyl to the tissue culture medium, seal the dish with parafilm,
Co-culture was carried out at 3 ° C in the dark for 3 days.

After co-cultivation, hypocotyls were transplanted to a callus induction medium containing carbenicillin 500 mg / L (40 hypocotyls / dishes) for eradication of Agrobacterium microorganisms. After that, the petri dish was sealed with surgical tape. 25
The cells were cultured for 7 days at 45 ° C, 45-55 µE and 16-day photoperiod.

The hypocotyls were transplanted to an adventitious bud formation medium containing carbenicillin 500 mg / L and kanamycin 10 mg / L (30 hypocotyls / dishes), and cultured for 2 weeks at 25 ° C., 45-55 μE and 16-day photoperiod. Until the adventitious buds were formed, the hypocotyls were subcultured every two weeks in an adventitious bud forming medium containing carbenicillin 500 mg / L and kanamycin 10 mg / L. Kanamycin resistant shoots formed in about 6-8 weeks.

The obtained kanamycin-resistant shoots were transplanted to an adventitious bud maturation medium containing carbenicillin 500 mg / L and kanamycin 50 mg / L. 25 ° C, 45-55 µE, 1
The cells were cultured under a 6-hour photoperiod for 3-4 weeks. Shoots in which the kanamycin resistance gene has not been introduced lose the white color.

The mature kanamycin resistant shoots were transplanted to an agripot containing a rooting medium containing carbenicillin 250 mg / L and kanamycin 50 mg / L to induce rooting. 3-4 at 25 ° C, 45-55μE, 16-hour photoperiod
Cultured for a week. A well-rooted seedling is transplanted to a pot (vermiculite: nursery soil 1: 1). It was sprinkled with sterile water and hung in a plastic bag. 20 ° C, 45-
The cells were grown at 55 μE under a 12-hour photoperiod. I gradually made a hole in the plastic bag and acclimated it.

[Example 2] In the same manner as in Example 1, transgenic plants were prepared for Tsukena variety friendly vegetables, red vegetable moss, turnip variety Hon Beni Daimaru turnip, and Japanese vegetable turnip. Table 4 shows the production rate of each variety.

[0045]

[Table 4]

Reference Example 1 The introduced GUS gene and the number of introduced genes in the transformants were examined by Southern blot analysis using the GUS gene as a probe. Leaves were collected from 6 individuals of the komatsuna variety “Osome” obtained in Example 1, and Murray and thompson (1980) Nucl. Acids
DNA was extracted by the method described in Res. 8: 4321-4325.
Then, the DNA was treated with a restriction enzyme BamHI or HindIII and subjected to Southern hybridization using the GUS gene as a probe.

FIG. 2 shows the case of treatment with BamHI and FIG. 3 shows the case of treatment with HindIII. In the figure, C indicates the non-transformant "offer" (control), and 1 to 6 indicate the individual numbers of the transformant "offer". As shown in FIG. 2, the GUS gene fragment (4.
(0kb) was confirmed. Moreover, as shown in FIG. 3, the number of integrated genes was 1-3 and varied depending on individuals. These transformants showed normal growth and were morphologically similar to the seed-derived control variety (FIG. 4). Moreover, pollen fertility was normal and self-fertilized seeds could be obtained.

[Reference Example 2] Agrobacterium belonging to the genus Agrobacterium exhibits a high gene transfer rate in plants of the Solanaceae and Cucurbitaceae family, but it is generally low in plants of the Brassicaceae family. Use of Um strain is desired. Therefore, the gene transfer rate was investigated using 5 strains of Agrobacterium. A feeder cell (tobacco [BY-2]) was added to the same hypocotyl as in Test Example 1,
The tissue was placed on a tissue culture medium whose pH was adjusted to 5.2 and precultured for 1 day.

On the other hand, LBA4404 (pLBA4404), C58C1Rif (pM
90), C58C1Rif (pGV2260), CIB542 / A136 (pCIB542),
Five strains of EHA101 (pEHA101), which belong to the genus Agrobacterium, were cultured overnight in a YBE liquid medium, and the obtained culture solution was diluted 10-fold with a tissue culture medium to prepare an infection solution. The pre-cultured hypocotyls were immersed in these five types of infection solutions for 10 minutes, returned to the tissue piece culture medium, and co-cultured at 25 ° C. for 2 days. Test Example 1 of hypocotyl GUS activity after co-culture
Same as above. Table 5 shows the results.

[0050]

[Table 5]

As shown in Table 2, CIB542 / A136, EHA10
1 showed high gene transfer rates of 45% and 48.5%, respectively. Both of these strains have a Ti plasmid derived from the Agrobacterium wild strain A281,
It exhibits a wide host range. No infection was observed with LBA4404, which is commonly used in Solanaceae and Cucurbitaceae.

[0052]

INDUSTRIAL APPLICABILITY The present invention provides a method for efficiently introducing a gene into a plant of the genus Brassica using a microorganism of the genus Agrobacterium to produce a transformed plant.

[Brief description of drawings]

FIG. 1 is a diagram showing the T-DNA region of the binary vector pIG121Hm.

FIG. 2 shows the results of Southern blotting of DNA treated with BamHI.

FIG. 3 is a diagram showing the results of Southern blotting of DNA treated with HindIII.

FIG. 4 is a photograph showing the morphology of a transformed plant organism.

[Procedure amendment]

[Submission date] April 1, 1996

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] Fig. 4

[Correction method] Change

[Correction contents]

FIG. 4

Claims (3)

[Claims] 1. A tissue piece of a Brassica plant is pre-cultured in a tissue culture medium, then immersed in an infection solution containing a microorganism of the genus Agrobacterium, and the tissue piece after the immersion coexists in the tissue culture medium. A method for introducing a gene into a plant of the genus Brassica by culturing, wherein the pH of the tissue piece culture medium is set to 5.0 to 6.9. 2. A tissue piece of a Brassica plant is pre-cultured in a tissue culture medium, then immersed in an infection solution containing a microorganism of the genus Agrobacterium, and the tissue piece after immersion coexists in the tissue culture medium. A method for introducing a gene into a plant of the genus Brassica by culturing, wherein the tissue piece culture medium contains a feeder cell. 3. A brassica plant tissue piece is pre-cultured in a tissue culture medium, then immersed in an infection solution containing a microorganism of the genus Agrobacterium, and the tissue piece after immersion coexists in the tissue culture medium. In a method for producing a transformed plant of the genus Brassica by culturing and then inducing callus from the tissue piece after co-culturing and redifferentiating the obtained callus, the following formula 82 <x / 10 + (y -19) ² + z <145 [where x is the time (minutes) of immersion in the infection solution, y is the temperature (° C) during co-culturing, and z is the period (hours) during co-culturing] x, y , Z is set, and a method for producing a transgenic plant of the genus Brassica is characterized by being set.