JP2002315460A - Method for making peripheral chimer and the chimera - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a versatile technique comprising surely making a peripheral chimera using a gene recombinant technique. SOLUTION: The method for making a peripheral chimera comprises transferring a plant cell with a vector for transferring a plant with a gene and then redifferentiating a plant body from an adventitious root obtained from the plant cell. The vector includes the aimed gene, rol gene as the selection marker gene, and an leavable DNA. The rol gene exists at a position where it makes an identical behavior with that of the leavable DNA, and the aimed gene exists at such a position as to express after leaving of the rol gene and the DNA factor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for reliably producing a peripheral chimera by a genetic engineering technique, and a peripheral chimera produced by this method.

[0002]

2. Description of the Related Art Synthetic peripheral chimeras of tomato are grafted by a tissue transplantation method, and are produced from adventitious buds composed of a fusion tissue generated by BA treatment of a healing cut surface. In citrus, a technique has been established for artificially producing a synthetic peripheral chimera by inducing adventitious buds from a grafted union.
Branching by mutation is widely used in chrysanthemum, and it is said that many branching varieties of chrysanthemum have a peripheral chimera structure. The mulberry line “FRM-01” grown by performing colchicine treatment on the mulberry cultivar “Kaneo” is a mixture of stable two- and four-medium peripheral chimeras. Therefore, it is considered that it is suitable for fruit collection and can be used for cultivation of a mulberry line for fruit collection with high productivity. In the 20th century, a new cultivar of Japanese pear, the peripheral chimera produced by gamma irradiation, was important for the development of disease resistance against black spot.

[0003]

The technique of producing a peripheral chimera of a plant is industrially important, but the probability of producing a peripheral chimera by a conventional method is very low and it takes a long time. Furthermore, the conventional method is only possible for plants in which techniques such as grafting have been established, and is not versatile. Garden crops,
If a technique for producing a reliable peripheral chimera that is versatile for crops, fruit trees, forest trees, and the like is developed, it is possible to make up for the defects of varieties with other varieties, and to improve the efficiency of breed improvement.

[0004] There have been reports of the formation of marginal chimeras by genetic recombination, but only suggest the possibility.

[0005] Accordingly, an object of the present invention is to provide a versatile technique for reliably producing a peripheral chimera using a gene recombination technique.

[0006]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a peripheral chimera can be prepared by using a vector containing a rol gene and an elimination factor, and completed the present invention.

Namely, a method of creating peripheral chimeras of the present invention comprises a DNA element having the target gene, rol gene, and de Hanareno as a selectable marker gene, and, ro
The l gene is located at a position where it behaves in unison with the detachable DNA element, and the target gene is located at such a position that the rol gene and the detachable DNA element are expressed after detachment. Present in the process of introducing a vector for gene transfer into plants into plant cells, culturing plant cells transfected with this vector, detecting adventitious roots generated during the culture and selecting this, and The process consists of cultivating the adventitious roots thus selected and redifferentiating the plant body.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

[0009] Here, the peripheral chimera refers to L1 and L2 of a plant.
And L3, which is a chimeric structure in which each layer is composed of genotypically or cytoplasmically different cell layers, and a peripheral chimera means a plant having such a chimeric structure.

[0010] The rol genes used as selectable marker gene in the present invention, gene present on the T-DNA of Agrobacterium rhizogenes, namely, rol
A, it is one or more genes selected from the rol genes consisting of rol genes containing rolB and rolC.

The target gene which can be introduced into a plant according to the present invention is not necessarily a gene capable of imparting an agriculturally superior trait, and is not necessarily imparting an agriculturally superior trait, but is required for basic research on a gene expression mechanism and the like. Various genes can be selected depending on the purpose.

[0012] The term "DNA element having detachment ability" refers to a DNA sequence capable of detaching itself from chromosomal DNA or the like in which these exist and function. In plants, such a factor is known as a transposon present on the chromosome, its structure and function,
And its behavior is almost completely known. In other words, in order for the transposon to function, in principle, it is expressed from a gene inside it and catalyzes its own elimination and transfer (transferase), and it is also present in its internal terminal region. Two components are required: the DNA sequence to which the transferase binds and acts. Due to these actions, the transposon is detached from the chromosome where it is present, and then usually translocates to a new position on the DNA, but sometimes loses its function without a certain probability of transposition and loses its function. Therefore, the present invention utilizes such transposon transposition errors. The transposon has such an autonomous transposon, namely, a transferase and a DNA-binding sequence, and the transferase expressed from the inside of the transposon binds to the DNA sequence existing in the terminal region and acts. By doing so, in addition to those that can autonomously detach from the chromosome where they exist and transfer, there is also a type called a non-autonomous transposon. This non-autonomous transposon is an autonomous transposon that, while retaining the DNA sequence at the end where the transferase binds and acts, has a mutation in the internal transferase gene and no expression of the transferase. Non-autonomous transposons cannot be detached from the chromosome.However, non-autonomous transposons are also referred to as autonomous transposons when a transferase is supplied from an autonomous transposon or a transferase gene that exists independently of this. It will show similar behavior.

At present, autonomous transposons isolated include Ac and Spm isolated from corn.
Has been analyzed in detail (A. Gieri
and H. Saedler, Plant Mol. B
iol. 19:39, 1992). In particular, Ac
It can be obtained by cutting out the wx-m7 locus in the maize chromosome with the restriction enzyme Sau 3A (U. Behrenset et al., Mol. Gen.
Genet. , 194: 346, 1984), the most analyzed autonomous transposon among plant transposons, and its DN Genet. 198: 19, 1984) It is suitable as a DNA factor used in the present invention because it can be easily obtained by those skilled in the art. Examples of non-autonomous transposons include Ds and dSpm , each of which lacks the internal regions of Ac and Spm (H.-P.D. Have been isolated from many plants such as snapdragon and morning glory (for example, Y. Inagaki et a
l. , Plant Cell, 6: 375,199
4). Incidentally, it is known in many cases that these transposons exert their abilities and are detached and translocated even when introduced into the chromosome of a plant of a different kind from the plant from which they are derived (for example, B .Baker et
al. Proc. Natl. Acad. Sci. US
A, 83: 4844, 1986).

In the present invention, any of autonomous and non-autonomous transposons can be used.
When a non-autonomous transposon is used, a transferase gene obtained or synthesized from an autonomous transposon or the like may be inserted and used in addition to the rol gene.

Further, as a DNA element having an elimination ability that exists outside of a plant, a site-specific recombination system (site
-Specific recognition s
ystem) are known. This site-specific recombination system comprises a recombination site having a characteristic DNA sequence (corresponding to a DNA element having an elimination ability of the present invention), and a sequence which specifically binds to this DNA sequence and has a sequence of 2%. When present, it consists of two elements, an enzyme that catalyzes recombination between the sequences, and
When two NA sequences are present on the same DNA molecule at a certain interval in the same direction, a region sandwiched between them is detached from the DNA molecule (plasmid, chromosome, etc.), In addition, when this arrangement is present at two places facing each other, the behavior is that this region is inverted. In the present invention, the former elimination action is utilized, but such elimination / inversion inside the recombination site occurs as a result of so-called homologous recombination by a site-specific recombination system. This is the most different point from the mechanism using a transposon, which causes its detachment as a process of transposition. The gene encoding the recombinase does not necessarily need to be present on the same DNA molecule as the recombination site. It is known that inversion can occur (NL Craig, Annu.
Rev .. Genet. , 22:77, 1988).

At present, site-specific recombination systems use C-isolated from microorganisms such as phage, bacteria (for example, Escherichia coli) and yeast.
re / lox system, pSR1 system, FLP system, cer system, f
im system and the like are known (for a review, see NL Cr
aig, Annu. Rev .. Genet. , 22: 1
7, 1988), its presence in higher organisms is not yet known. However, the site-specific recombination system isolated from these microorganisms is also described in WO 93/0128 described above.
In Publication No. 3, Cre / lox derived from P1 phage
When a system is introduced into a different species (including a plant) from the species from which it is derived, as in the case of using a system for a gene transfer vector into a plant, the same behavior as that in the original organism is obtained. It is clear that it can be taken. In one embodiment of the present invention is the way, yeast (Zygos
The R / RS system (H. Matsuzuki ), which is a site-specific recombination system of S. accharomyces rouxii ).
et al. J. Bacteriology, 17
2: 610, 1990), with the use of a recombination enzyme inserted between its recombination sites, but this R / RS system has also been reported to maintain its original function in higher plants. (H. Onuchi et al, N
ucleic Acid Res. , 19: 6373,
1991).

In the present invention, a gene includes a promoter sequence and a terminator sequence essential for its expression. These can be used without limitation as long as they function in plants. For example,
Such promoters include the cauliflower mosaic virus 35S promoter (JT Odell).
et al. , Nature (London), 31
3: 810, 1985), the promoter of nopaline synthase (WHR Langridge et al.,
Plant Cell Rep. 4: 355, 198
5) and the like, and as a terminator, a polyadenylation signal of nopaline synthase (A. Depicker).
et al. J. Mol. Appl. Gen. ,
1: 561, 1982), polyadenylation signal of octopine synthase (J. Gilen et al.,
EMBO J.C. 3: 835, 1984) can be used.

Furthermore, if an inducible promoter is used,
Gene expression can be controlled. Many such inducible promoters are known to date. For example, as a substance that reacts with a chemical substance, a promoter of a glutathione-S-transferase I gene (Japanese Patent Laid-Open No.
268965), a promoter of a glutathione-S-transferase II gene (International Publication WO9
3/01294), a Tet repressor fusion type cauliflower mosaic virus 35S promoter (C.
Gatz et al. , Mol. Gen. Gene
t. 227: 229, 1991), Lac operator / repressor promoter (RJ Wilde).
et al. , The EMBO Journal,
11: 1251, 1992), an alcR / alcA promoter (International Publication WO94 / 03619),
Glucocorticoid-based promoter (Takashi Aoyama, Protein Nucleic Acid Enzyme, 41: 2559, 1996), par-based promoter (T. Sakai et al., Plant)
Cell Physiol. 37: 906,199.
6) and the like, and those that respond to light as ribulose diphosphate carboxylase small subunit gene (rbc
S) promoter (R. Fluhr et al.,
Proc. Natl. Acad. Sci. USA, 8
3: 2358, 1986), the promoter of the fructose-1,6-bisphosphatase gene (Tokuheihei 7-5).
No. 01921), a promoter of a light-harvesting chlorophyll a / b binding protein gene (Japanese Patent Application Laid-Open No. 5-89), and further, a promoter which is responsive to various external environments such as injury and temperature are known. .

In the present invention, the selection marker r
These promoters can be appropriately used to control the expression of the ol gene, the target gene, a DNA factor having detachment ability, and other genes.

[0020] The present invention provides a selection marker gene, ro
(1) a vector in which the l gene is placed at a position where it behaves together with a detachable DNA element, and a target gene is placed at a position where the rol gene and the detachable DNA element are expressed after detachment. Is used to introduce a gene into a plant. As long as such requirements are satisfied, the rol gene, the DNA element having the elimination ability and the target gene may be arranged in the same vector or may be present on different vectors.

The site for inserting the rol gene may be a site at which the rol gene can be eliminated together with the DNA element capable of being eliminated. For example, when a transposon is used as a DNA element having an elimination ability, it does not affect the elimination of the transposon upstream of the promoter region of the transferase gene and downstream of the terminal region to which this transferase binds. You can insert this in the position. When the R / RS system is used, it can be inserted anywhere as long as it does not inhibit the expression of the recombinase within the region between the recombination sites.

The target gene may be located, for example, between its promoter and the structural gene. By arranging the target gene in this manner, the target gene is expressed after the roll gene and the DNA factor having the elimination ability are eliminated.

The gene can be introduced by indirectly introducing the vector into a plant cell via a virus or a bacterium which infects a plant (I. Potryku).
s, Annu. Rev .. Plant Physiol.
Plant Mol. Biol. , 42: 205, 19
91). In this case, for example, cauliflower mosaic virus, gemini virus, tobacco mosaic virus, brom mosaic virus, and the like can be used as the virus, and bacteria such as Agrobacterium tumefaciens (hereinafter abbreviated as A. tumefaciens) and Agrobacteria can be used. Um rhizogenes (hereinafter abbreviated as A. rhizogenes) and the like can be used. In general, Agrobacterium does not infect monocotyledonous plants, but only dicotyledonous plants.Recently, there have been reports of cases in which these are transmitted to monocotyledonous plants and transfected. (For example, International Publication WO94 / 00977).

[0024] Gene transfer can also be carried out by subjecting this vector to microinjection, electroporation,
It can also be carried out by direct introduction into plant cells by a physical / chemical method such as a polyethylene glycol method, a fusion method, and a high-speed ballistic penetration method (I. Potrykus, Annu. Rev. Plan).
tPhysiol. Plant Mol. Biol. ,
42: 205, 1991). For many monocotyledonous plants and dicotyledonous plants that are not easily infected by Agrobacterium, the direct introduction method using Agrobacterium, which is widely used for gene transfer, cannot be used. It is.

In the present invention, since the rol gene derived from Agrobacterium is used as a selection marker gene, adventitious roots differentiate from cells into which the gene has been introduced. Such adventitious roots can be regenerated from plants by culturing each plant under known conditions. During the regeneration process, the L
In the three layers, among the target gene, the rol gene, and the detachable DNA factor introduced into the plant as described above, the rol gene and the detachable DNA factor are detached, and the target gene is expressed. However, in the L1 and L2 layers of the same plant tissue, such detachment does not occur, so that the target gene is not expressed in these tissues. That is, only in the L3 layer, the selectable marker gene is removed, and a peripheral chimera expressing the target gene is created.

[0026]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The details of the present invention will be described below with reference to embodiments.
However, the present invention is not limited to the following examples. In the following examples, more detailed experimental procedures for molecular biological techniques are described in Molecular Cloning (Sambr) unless otherwise specified.
OK et al. , 1989) or according to the manufacturer's instructions.

[0027] [Example 1] I. Preparation of Vector Plasmid p35Rint (Japanese Patent Laid-Open No. 2000-3422)
With the restriction enzymes Eco RI and Hind III 6) cut out Rint gene linked to the cauliflower mosaic virus 35S promoter, also restriction enzymes Ec
o pNPI128 treated with RI and Hind III
(Japanese Unexamined Patent Application Publication No. 9-154580), and the plasmid pR
S35Rint was obtained (FIG. 1).

Next, in order to eliminate the SseI restriction enzyme site of the rol gene, pBluescriptIISK was used.
+ Cut rol genes are cloned in (Toyobo (purchased from Ltd.)) a (JP-A 2000-34226) by restriction enzyme Sse I, and ligated with blunt-ended with T4DNA polymerase pSKrolΔss
e was obtained. (FIG. 2). Moreover, cut out rol delta sse gene fragment with a restriction enzyme Eco RI from PSKRolderutasse, it was inserted into Eco RI restriction enzyme sites PRS35Rint, to obtain a plasmid PRS35RintRol (Figure 3).

The target vector is pRS35Rin
From tRol, Rin linked to the rol Δ sse gene and the cauliflower mosaic virus 35S promoter
t gene and the Rs in these ends, restriction enzyme S
It was cut out with se I and pNP105 (Japanese Patent Laid-Open No. 2000-2000)
Obtained by inserting the Sse I restriction enzyme site of 34,226), which was designated as plasmid PEXM120 (Figure 4).

The obtained plasmid pEXM120 was introduced into Escherichia coli DH5α strain. coli D
H5α (pEXM120) as of March 28, 2001
It was attached to the domestic deposit by date.

II. PEXM1 for Agrobacterium
Introduction of A.20 Tumefaciens LBA4404 strain was added to 10 ml of YEB liquid medium (beef extract 5 g / l, yeast extract 1).
g / l, peptone 1 g / l, sucrose 5 g / l, 2 mM
gSO4, pH 7.2 at 22 ° C (hereinafter referred to as the pH at 22 ° C unless otherwise specified), and OD63.
The cells were cultured at 28 ° C. until 0 was in the range of 0.4 to 0.6. The culture was centrifuged at 6900 × g at 4 ° C. for 10 minutes to collect the cells, and the cells were collected in 20 ml of 10 mM HEPES.
(PH 8.0), and again 6900 × g at 4 ° C.
The cells are collected by centrifugation for 10 minutes, and the cells
Was suspended in a YEB liquid medium and used as a bacterial solution for plasmid introduction. In a 0.5 ml tube, 50 μl of the plasmid-introducing bacterial solution and 3 μl of the plasmid pEXM120 were mixed, and the resulting mixture was electroporated (Gene Pulser II system [BIORAD]) to introduce the plasmid, and then 200 μl of the YEB liquid The medium was added, and the cells were cultured by shaking at 25 ° C. for 1 hour. This cell is
YEB agar medium supplemented with 0 mg / l kanamycin (agar 1.
5% w / v, other composition is the same as above. 28)
The cells were cultured at 2 ° C. for 2 days to obtain bacterial colonies. Further, after transplanting this bacterial colony into a YEB liquid medium and further culturing, a plasmid was extracted from the bacterial by an alkaline method, and The plasmid pEXM12 was added to the Tumefaciens strain LBA4404.
0 was introduced, and the bacterium was transferred to LBA44.
04 (pEXM120).

III. LBA4404 to tobacco (pE
XM120) and preparation of transformant YEB agar medium (10 g / l bactopeptone, 10 g / l)
Agrobacterium LBA4404 (pEXM120) cultured with 1 bacto yeast extract, 5 g / l sodium chloride, 15 g / l bacto agar) was prepared using YEB.
After culturing overnight in a liquid medium at 25 ° C. and 180 rpm, YEB
Suspended in a liquid medium so that OD ₆₀₀ = 0.25,
An Agrobacterium suspension for infection was used.

Tobacco grown in a greenhouse ( Nicoti)
ana tabacum cv. SR1, the same applies hereinafter unless otherwise specified. ) Was sterilized by immersion in a 1 v / v% aqueous solution of sodium hypochlorite for 5 minutes, washed three times with sterilized water, and then the middle vein was removed to prepare leaf pieces of about 8 mm square. The tobacco leaf pieces were obtained from A. coli having introduced pEXM120 in II. Tsubame Science LBA
After immersion in the Agrobacterium suspension of strain 4404 for about 1 minute to infect it, put it on sterile filter paper to remove excess bacterial solution, and then use acetosyringone 50 mg / l.
Contains no plant hormones (hormone free)
MS agar medium (T. Murashigeand FS)
kool, Physiol. Plant. , 15:47
3, 1962, with the addition of agar 0.8w / v%)
The leaves were placed so that the backs of the leaves were facing up. This was cultured for 2 days in the dark at 25 ° C., transplanted to a hormone-free MS agar medium containing only 250 mg / l ticarcillin, and continued to be cultured for 3 weeks. As a result, adventitious roots were obtained. These adventitious roots were separated and cultured in a medium having the same composition for an additional week. As a result, 11 lines in which the adventitious roots elongated and diverged vigorously were selected, and B
The cells were cultured on an MS agar medium containing 1.0 mg / l of A, 0.1 mg / l of NAA, and 250 mg / l of ticarcillin, and 40 adventitious buds were obtained from 11 lines of adventitious roots.
These individuals were cut off from adventitious roots, rooted on an MS medium containing 250 mg / l ticarcillin, subcultured every month, and subjected to the following analysis.

IV. 35S detection pEXM120 desorption of DNA factor having a rol genes and de Hanareno by GUS staining when recombinase desorption of DNA factors and rol genes having a leaving Hanareno occurs worked, inserted as a model of useful genes -Designed to express the GUS gene. That is, in this case, detachment of the cassette can be detected by expression of 35S-GUS. Therefore, a part of the plant obtained in III is cut out, and the method of Jefferson et al. (Plant Mo
l. Biol. Rep. , Vol. 5: 387, 198
When a GUS activity test was carried out in accordance with 7), GUS activity was confirmed in 25 of the 37 strains, and it was revealed that the DNA factor and rol gene capable of leaving were removed from the plant chromosome. Was.

V. DN with rol genes and de Hanareno by PCR analysis GUS staining
Chromosome D for 25 individuals in which factor A detachment was observed
NA is used as Fast DNA Kit (BIO 101 I
nc. ), And PCR analysis was performed to confirm that the region between the chromosomes and the RS containing the rol gene was detached.

On the other hand, using primers designed so rol genes introduced is amplified was subjected to detection of rol genes, rol genes were amplified in 17 individual, also rol gene in these individuals also It turned out to be present.

From the results of the above-mentioned GUS staining and PCR analysis, these individuals show that the cells from which the rol gene and the DNA factor having the elimination ability have been removed by elimination and the cells remaining on the chromosome without removing these elements. It was suggested that this was a chimera individual consisting of cells that had been used.

V. Tissue Observation by Microscope Plants suggested to be chimeras were adapted to two strains,
Cultivated for about 2 months under sunlight. Using leaves and shoot tips of these plants as materials, a micro slicer (Osa
After preparing a section having a thickness of 70 micrometers by using Ka EM, DTK1000), a GUS activity test was performed in the same manner as described above, and the state of GUS staining was observed with a microscope. In these individuals, it was confirmed by PCR analysis that the rol gene and the DNA element having the elimination ability were simultaneously removed and the presence of the rol gene was confirmed. The detachment of the rol gene and the detachable DNA factor has also been observed by GUS staining. As a result of observation with a microscope, L3
GUS staining was confirmed only in the layer, and it was revealed that the L3 layer peripheral chimera was prepared.

The results are shown in FIGS. FIG. 5 is a photograph of a leaf section. The portion stained blue by GUS staining is the L3 layer. FIG. 6 is a photograph of a cross section of the shoot apex portion. The part stained blue by GUS staining is L3
Layer.

[0040]

According to the present invention, a peripheral chimera can be reliably produced by a genetic engineering technique. Therefore, the present invention enables plant breeding by genetic engineering techniques.

[0041]

[Brief description of the drawings]

FIG. 1 pRS3 of the pEXM120 creation scheme
FIG. 5 is a diagram showing up to creation of 5Rint.

FIG. 2 pSKr of the pEXM120 creation scheme
FIG. 6 is a diagram showing up to creation of olΔsse.

FIG. 3 pRS3 of the pEXM120 creation scheme
5Rint and pSKrollΔsse to pRS35R
FIG. 9 is a diagram showing up to creation of intRol.

FIG. 4 pRS3 of the pEXM120 creation scheme
FIG. 5 is a diagram illustrating a process from 5RintRol to creation of pEXM120.

FIG. 5 shows a GUS of a leaf section of pEXM120-introduced tobacco.
It is a photograph showing the situation at the time of dyeing.

FIG. 6 shows the cross section of the shoot tip of pEXM120-introduced tobacco by GU.
It is a photograph showing the situation at the time of S dyeing.

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] October 12, 2001 (2001.10.
12)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0030

[Correction method] Change

[Correction contents]

The obtained plasmid pEXM120 was introduced into Escherichia coli DH5α strain. coli D
H5α (pEXM120) was deposited in Japan (accession number: FERM P-18280).

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Akiyoshi Kawaoka 5-21-1, Oji, Kita-ku, Tokyo Nippon Paper Industries Co., Ltd. (72) Inventor Hiroyasu Ebinuma 5-21, Oji, Kita-ku, Tokyo No. 1 Nippon Paper Industries Co., Ltd. Technical Research Laboratory F-term (reference) 2B030 CA15 CA17 CA19 4B024 AA08 CA04 DA01 EA04 FA02 FA10 GA11

Claims (4)

[Claims] 1. A method for producing a peripheral chimera, which is carried out through the following steps (A), (B) and (C). (A) a target gene, comprising a DNA factor having a rol gene, and de Hanareno as a selectable marker gene, and, r
The ol gene is located at a position where it behaves in unison with the detachable DNA element, and the target gene is located at a position where it is expressed after the rol gene and the detachable DNA element are detached. (B) A step of culturing plant cells transfected with this vector, detecting adventitious roots generated during the culturing, and selecting the same ( C) A process of culturing the adventitious root selected in (B) and redifferentiating the plant body 2. A detachable DNA element and / or rol between a promoter of a target gene and a structural gene.
The method for producing a peripheral chimera according to claim 1, wherein a vector for introducing a gene into a plant into which the gene has been inserted is used. 3. A peripheral chimera produced by the following steps (A), (B) and (C). (A) a target gene, comprising a DNA factor having a rol gene, and de Hanareno as a selectable marker gene, and, r
The ol gene is located at a position where it behaves in unison with the detachable DNA element, and the target gene is located at a position where it is expressed after the rol gene and the detachable DNA element are detached. (B) A step of culturing plant cells transfected with this vector, detecting adventitious roots generated during the culturing, and selecting the same ( C) A process of culturing the adventitious root selected in (B) and redifferentiating the plant body 4. A detachable DNA element and / or rol between a promoter of a target gene and a structural gene.
The peripheral chimera according to claim 3, which is prepared using a vector for introducing a gene into a plant into which the gene has been inserted.