JP2000342253A - Improvement in efficiency of gene transfer to plant cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the efficiency of gene transfer to a plant cell simply carried out through a bacterium of the genus Agrobacterium without damaging a tissue and to better a breed by heat-treating and centrifuging a plant cell, etc. SOLUTION: A plant cell or a plant tissue of rice plant, maize, lawn grass, etc., is heat-treated at 33-60 deg.C, preferably 35-55 deg.C, more preferably 37-52 deg.C for 5 seconds to 24 hours and centrifuged at 100-250,000 G, preferably 500-200,000 G, more preferably 1,000-150,000 G centrifugal acceleration for 1 second to 4 hours to improve the efficiency of gene transfer to a plant cell carried out through a bacterium of the genus Agrobacterium. Preferably after the plant cell or plant tissue is heat-treated, a gene transfer treatment is performed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for improving the efficiency of gene transfer into plant cells.

[0002]

2. Description of the Related Art Agrobacterium-based transformation methods are generally efficient, have a low copy number of the gene to be introduced, and can be introduced without fragmenting a specific region called T-DNA in a short time. It has many excellent features, such as the fact that a transformant can be obtained by cultivation of E. coli. For this reason, it is widely used as the most useful transformation means in various plant species.

[0003] As described above, the Agrobacterium method is a very excellent method for transforming plants, but the success and efficiency of transformation vary greatly depending on the plant species, genotype, and plant tissues used. It is a fact (Potrykus
et al. 1998 (references (36)). That is, there are plant species that have not been successfully transformed, and many plant species that can be transformed by only a small number of varieties. Also, some plant species have limited available tissues and cannot handle large amounts of material. In order to produce practical varieties by genetic recombination, it is necessary to produce a large number of transformed plants and then select a line having the desired trait. However, the types of crops from which a large number of transformants can be easily obtained for this purpose are currently limited to some. Therefore, there is a strong demand for the development of an improved method that can solve such problems.

[0004] The Agrobacterium-mediated transformation method itself varies the composition of a test material or a culture medium used for cultivation depending on the plant species, but contacts a tissue of the material with a suspension of Agrobacterium. The procedure of selecting transformed cells after co-culture and producing transformed plants is almost common. Agrobacterium is usually transmitted to plant tissue as a material without sterilization as necessary, but without any other special treatment (Rogers et al. 1988 (Reference (37)). )), Visser 199
1 (Reference (41)), McCormick 1991 (Reference (31)), Lin
dsey et al. 1991 (references (30))). Therefore, research on the improvement of the transformation system has been conducted mainly on the Agrobacterium strain, the vector composition, the medium composition, the type of the selectable marker gene and promoter, the type of the test tissue, and the like.

[0005] In contrast, few studies have been conducted on the concept of converting plant tissue before inoculation with Agrobacterium into a physiological state in which gene transfer is likely to occur. If it can be converted to such a physiological state by some simple treatment, it will be very useful, and in addition to improving the gene transfer efficiency, it will make it possible to transform plant species and genotypes, which were difficult in the past. Is also expected to be effective. Examples of previous studies on plant tissue pretreatment include particle gun (Bidney et al., 1992).
(Reference (6))) and ultrasound (Trick HN et al., 1997
(Ref. (40))). Both aim to physically injure the tissue, thereby promoting the invasion of bacteria into plant tissue and increasing the number of plant cells to be infected. However, this is only a development of the leaf disk method (Horsch et al., 1985 (reference (19))) which has been widely used, and is not a processing method based on a new concept. The degree of effect and versatility are not clear, and at present it is not used as a general method.

[0006]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a plant which can carry out gene transfer easily and without damaging the tissue with higher efficiency than the conventional gene transfer method using the Agrobacterium method. An object of the present invention is to provide a method for improving the efficiency of gene transfer into cells.

[0007]

Means for Solving the Problems As a result of earnest studies, the present inventors have found that in a gene transfer method using Agrobacterium, a plant cell or plant tissue to be subjected to gene transfer is subjected to heat treatment and centrifugation. The present inventors have found that the gene transfer efficiency can be significantly improved, and completed the present invention.

That is, the present invention provides a method for improving the efficiency of gene transfer into plant cells via Agrobacterium bacterium, which involves heat treatment and centrifugation of plant cells or plant tissues.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the method of the present invention, a method for introducing a gene through a bacterium of the genus Agrobacterium involves heat treatment and centrifugation of a plant cell or a plant tissue into which the gene is to be introduced. After heat treatment and centrifugation, the plant cell or plant tissue may be brought into contact with the Agrobacterium bacterium at room temperature and under normal gravity, or may be brought into contact with the Agrobacterium bacterium while performing the heat treatment and / or centrifugation. You may. In the case where heat treatment and centrifugation treatment are performed before contact with Agrobacterium bacteria, these treatments may be performed simultaneously, or one of the treatments may be performed first and then the other may be performed. .

The heat treatment conditions are appropriately selected according to the type of plant to be used and the amount of cells or tissues to be heat-treated, but are usually 30 ° C. to 60 ° C., preferably 33 ° C. to 55 ° C., more preferably 37 ° C. This is performed in a temperature range of about -52 ° C. The heat treatment time is appropriately selected depending on the heat treatment temperature, the type of plant to be used, the type of cells or tissues to be heat-treated, and the like, but is usually about 5 seconds to 24 hours. In addition, even if the heat treatment time is short when the heat treatment temperature is high, the gene transfer efficiency can be significantly improved. For example, when the heat treatment temperature is 60 ° C., the gene transfer efficiency can be significantly improved even with a heat treatment time of about 5 seconds. On the other hand, when the heat treatment temperature is as low as about 34 ° C., gene transfer efficiency can be significantly improved by heat treatment for several tens of hours. Particularly preferred heat treatment conditions are usually from 37 ° C. to 52 ° C. for about 1 minute to 24 hours, but appropriate heat treatment conditions for the plant cells or plant tissues can be easily set by routine experiments. When the plant cells or the plant tissues are heat-treated at a temperature of 55 ° C. or higher for a long time, the plant cells may be damaged and the transformation efficiency may decrease. It is preferable to shorten the time, for example, to about 3 minutes or less, preferably about 1 minute or less so that the plant cells are not damaged.

The conditions for centrifugation are appropriately selected according to the type of plant used and the like, but are usually 100 G to 250,000 G, preferably 500 G to 200,000 G, and more preferably 100 G to 200,000 G.
This is performed in a centrifugal acceleration range of about 0 G to 150,000 G. The time for the centrifugation treatment is appropriately selected depending on the centrifugal acceleration, the type of plant to be used, and the like. There is no particular upper limit on the centrifugation time, but the object can usually be achieved in about 10 minutes. The centrifugation time can be significantly shortened when the centrifugal acceleration is large, for example, 1 second or less, to significantly improve the gene transfer efficiency. On the other hand, when the centrifugal acceleration is small, gene transfer efficiency can be significantly improved by performing the centrifugal treatment for a long time. Particularly preferred centrifugation conditions are 500 G to 200,000 G, particularly 1,000 G to
Although it is often about 1 second to 2 hours at 150,000 G,
Appropriate centrifugation conditions for the plant cell or plant tissue can be easily set by routine experimentation.

The method of the present invention uses a plant cell or a plant tissue which has been subjected to heat treatment and centrifugation treatment as a plant cell or plant tissue to be contacted with Agrobacterium genus bacteria, or contacts the Agrobacterium bacterium with heat treatment and centrifugation treatment. As a method for gene introduction or transformation using an Agrobacterium bacterium, a well-known method can be applied as it is.

[0013] A method for introducing or transforming a gene into a plant using Agrobacterium bacteria is well known in the art and widely used.

Agrobacterium (Agrobacterium)
ium tumefaciens has been found in many dicotyledonous plants to have root carcinoma (c)
It has long been known to cause rown gall disease, and in the 1970s it was discovered that the Ti plasmid was involved in virulence and that T-DNA, a part of the Ti plasmid, was integrated into the plant genome. Was done. Then this
T-DNA has been found to contain genes involved in the synthesis of hormones (cytokinins and auxins) required for carcinogenesis. Excision of T-DNA and transmission to plants require genes present in the virulence region (vir region) on the Ti plasmid, and T-DNA is required for excision of T-DNA.
-A border sequence at both ends of the DNA is required. Agrobacterium rhizog, another Agrobacterium bacterium
enes also has a similar system with the Ri plasmid (FIGS. 3 and 4).

Agrobacterium infection causes T-DNA
Is integrated into the plant genome, so it was expected that this gene would also be integrated into the plant genome when a desired gene was inserted into T-DNA. However, the Ti plasmid was
Due to the size as large as 0 kb or more, it was difficult to insert a gene into T-DNA on a plasmid using standard genetic engineering techniques. Therefore, a method for inserting a foreign gene on T-DNA was developed.

First, LBA4404 (Hoekema et al., Supra), which is a disarmed strain in which the hormone synthesis gene has been removed from the T-DNA of the neoplastic Ti plasmid.
1983 (Reference (14))), C58C1 (pGV3850) (Zambryski et
al., 1983 (Reference (44))), GV3Ti11SE (Fraley et al.,
1985 (reference (10))) and the like (FIG. 3). By using these, two methods have been developed for introducing a desired gene into T-DNA of Agrobacterium Ti plasmid or introducing T-DNA having the desired gene into Agrobacterium. One of them is to use an intermediate vector, which is easy to genetically manipulate, can insert the desired gene, and can be replicated in E. coli, into the T-DNA region of the disarmed Ti plasmid of Agrobacterium. Crossing method (t
riparental mating) (Ditta et al., 1980 (references)
(9) is a method of introducing by homologous recombination via)),
Called the intermediate vector method (Fraley et al., 1985 (ref. (10)); Fraley et al., 1983 (ref. (11)); Zamb
ryski et al., 1983 (Reference (44)), JP-A-59-140885
No. (EP116718)). The other is a binary vector (b
(Fig. 3), which shows that the integration of T-DNA into plants requires the vir region but does not need to be on the same plasmid to function (Hoekema et al. ., 1983 (references (14)). The vir region includes virA, virB, virC, virD, virE, and virG. (Encyclopedia of Plant Biotechnology (published by Enterprises, Inc. (1989))), the vir region is defined as virA, virB, virC, virD, It includes all of virE and virG. Therefore, the binary vector is T-DN
A in which A is incorporated into a small plasmid that can be replicated in both Agrobacterium and Escherichia coli. This is introduced into Agrobacterium having a disarmed Ti plasmid and used. The introduction of the binary vector into Agrobacterium can be performed by a method such as an electroporation method or a three-way hybridization method. Binary vectors include pBIN19 (Bevan, 1984 (reference (5))), pBI121
(Jefferson, 1987 (Ref. (21))), pGA482 (An et al.,
1988 (Reference (2)), JP-A-60-70080 (EP120516))
Many new binary vectors have been constructed based on these and used for transformation. In the Ri plasmid system, similar vectors have been constructed and used for transformation.

Agrobacterium A281 (Watson et al.,
1975 (reference (42))) is strongly pathogenic (super-virulent)
Strain, its host range is wide, and the transformation efficiency is higher than other strains (Hood et al., 1987 (Ref. (15)); Kom
ari, 1989 (references (23)). This property has A281
Ti plasmid pTiBo542 (Hood et a
l., 1984 (ref. (18)); Jin et al., 1987 (ref.
(22)); Komari et al., 1986 (references (26))).

Two new systems have been developed using pTiBo542. One is strain EHA101 (Hood et a) harboring the disarmed Ti plasmid of pTiBo542.
l., 1986 (Ref. (17))) and EHA105 (Hood et al., 19
93 (reference (16))), and by applying these to the above-described binary vector system,
It is used for transformation of various plants as a system having high transformation ability. The other is a 'super-binary' vector (Hiei et al., 1994).
(Ref. (13)); Ishida et al., 1996 (Ref. (20));
Komari et al., 1999 (references (28)), WO 94/00977,
WO95 / 06722) system (FIG. 4). This system uses the vir domain (virA, virB, virC, virD, virE and virG
(Hereinafter, each of these may be referred to as a “vir fragment region”.)), Which is a kind of binary vector system since it is composed of a disarmed Ti plasmid and a plasmid having T-DNA. However, T-
A plasmid having a DNA, that is, a fragment of a vir region in which at least one vir fragment region has been substantially removed from a vir fragment region in a binary vector (preferably a fragment containing at least virB or virG, more preferably
fragment containing virB and virG) (Komari, 1990a
(Reference (24)) The difference is that a super binary vector is used. Agrobacterium having a super binary vector, T-
In order to introduce a DNA region, homologous recombination via the triple hybridization can be used as an easy method (Komari et al., 1996).
(Reference (27)). This superbinary vector system has been shown to provide very high transformation efficiencies in many plant species as compared to the various vector systems described above (Hiei et al., 1994 (13). ;
Ishida et al., 1996 (Ref. (20)); Komari, 1990b
(Ref. (25)); Li et al., 1996 (Ref. (29)); Sai
to et al., 1992 (ref. (38)).

In the method of the present invention, the host Agrobacterium is not particularly limited.
Agrobacterium tumefaciens (for example, Agrobacterium
iumtumefaciens LBA4404 (Hoekema et al., 1983 (Reference (14))) and EHA101 (Hood et al., 1986 (Reference (1)
7))) can be preferably used.

According to the method of the present invention, a significant effect can be obtained without any particular limitation as long as it is a gene transfer system based on the expression of genes in the pathogenic (vir) region in Agrobacterium bacteria. it can. Therefore, the present invention can be used for any vector system such as the above-mentioned intermediate vector, binary vector, strongly pathogenic binary vector, and super binary vector, and the effects of the present invention can be obtained. The same applies when different vector systems in which these vectors are modified are used (for example, Agrobacterium sp.
cutting out part or all of the vir region and additionally incorporating it into a plasmid, cutting out part or all of the vir region and introducing it into Agrobacterium as part of a new plasmid, etc.). Naturally, according to the method of the present invention, even in wild-type Agrobacterium bacteria,
The efficiency of introduction of a wild-type T-DNA region into a plant can be increased, and the infection efficiency can be effectively improved.

The desired gene to be introduced into the plant is
Based on a suitable selection marker such as a gene having resistance to a drug such as kanamycin or paromomycin, which can be incorporated into a restriction enzyme site in the T-DNA region of the above-described plasmid by a conventional method and simultaneously or separately incorporated into the plasmid. You can choose. For large ones having many restriction sites, it may not always be easy to introduce the desired DNA into the T-DNA region by ordinary subcloning techniques. In such a case, the target DNA can be introduced by the homologous recombination in the cells of the bacterium of the genus Agrobacterium by the three-way hybridization method.

Further, the plasmid was used for Agrobacterium tumefa
The operation of introducing into a bacterium belonging to the genus Agrobacterium such as ciens can be performed by a conventional method. Examples of the method include the above-mentioned three-way hybridization method, electroporation method, electroinjection method, and chemical treatment such as PEG. And so on.

The gene to be introduced into a plant is basically located between the left and right border sequences of T-DNA, as in the prior art. However, since the plasmid is circular, the number of boundary sequences may be one, and if a plurality of genes are to be arranged at different sites, there may be three or more boundary sequences. Also, in Agrobacterium bacteria, it may be located on a Ti or Ri plasmid or on another plasmid. Furthermore, it may be arranged on a plurality of types of plasmids.

The method of introducing a gene via Agrobacterium can be carried out by simply contacting a plant cell or plant tissue with Agrobacterium. For example, a suspension of Agrobacterium belonging to the genus Agrobacterium having a cell concentration of about 10 ⁶ to 10 ¹¹ cells / ml is prepared, and plant cells or plant tissues are immersed in the suspension for about 3 to 10 minutes. For several days.

The cells or tissues to be subjected to gene transfer are not particularly limited, and may be leaves, roots, stems, nuts, or any other site, or dedifferentiated cells such as callus. An embryo that has not been dedifferentiated may be used. Although the type of plant is not limited at all, angiosperms are preferred, and dicots or monocots may be used as long as they are angiosperms.

As specifically shown in the following Examples, the method of the present invention significantly improves the efficiency of gene transfer as compared with the conventional Agrobacterium method. In addition, the gene transfer efficiency of plants that could be transfected by the Agrobacterium method was improved, and this method was also applied to plants that could not be transfected by the conventional Agrobacterium method. The method of the invention has made gene transfer possible for the first time. Therefore, “improving the efficiency of gene transfer” in the present invention also includes enabling gene transfer that was not possible by the conventional method (ie, the gene transfer efficiency which was conventionally 0%). I think it improved).

[0027]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below more specifically based on embodiments. However, the present invention is not limited to the following examples.

Example 1 (1) Test Tissue and Test Bacterial System Morning light of Japonica rice was used as a test variety, and immature embryos were used as materials. Test immature embryos were collected from immature seeds 1 to 2 weeks after flowering and prepared by the method of Hiei, Y., et al. (Reference (13)). That is, after flowering, the immature seeds on the 7th to 12th days were removed from the immature seeds and then placed in 70% ethanol for 30 seconds.
After disinfection by immersion in 1% sodium hypochlorite for 10 minutes, immature embryos were taken out and used as test materials. Also,
The calli derived from immature embryos are immature embryos containing 4g / l Gelrite
Medium (Hiei et al. 1994 (reference (13)), (N6 inorganic salts and vitamins (Chu CC 1978 (reference (8)),
g / l casamino acid, 2 mg / l 2,4-D)) for 2 weeks.

As an Agrobacterium strain and a plasmid vector, LBA4404 (pIG121Hm) (Hiei et al., 199)
4 (Reference (13))), LBA4404 (pNB131) (see FIG. 2), LBA
4404 (pTOK233) (Hiei et al., 1994 (reference (13))) was used.

The construction of pNB131 was performed as follows. pS
After introducing B31 (Ishida Y, 1996 (Reference (20)) into the E. coli LE392 strain, pNB1 (Komari T et al., 1996 (Reference) was obtained by the Triparental mating method (Ditta G, 1980 (Reference (9))). (27)) and introduced into Agrobacterium strain LBA4404. Homologous recombination between pNB1 and pSB31 in Agrobacterium yielded pNB131.

The T-DNA region of pIG121Hm has a kanamycin resistance (nptII) gene controlled by a nopaline synthase (nos) gene promoter and a hygromycin resistance (hpt2) gene controlled by a cauliflower mosaic virus (CaMV) 35S promoter. ) Gene, a GUS gene controlled by the 35S promoter and mediated by an intron of the castor catalase gene (Ohta, S. et al., 1990 (Reference (3)
3)).

The T-DNA region of pNB131 has a bar gene controlled by the 35S promoter and a GUS gene controlled by the 35S promoter and mediated by an intron (described above).

The T-DNA region of pTOK233 contains the nptII gene controlled by the nos promoter, the hpt gene controlled by the 35S promoter, and the GUS gene controlled by the 35S promoter and mediated by introns (described above). pTOK233 is a super binary vector with high transformation ability (Komari, T. et al., 1999 (reference (2)
8))).

(2) Heat treatment Five to 200 mg of the test tissue was immersed in a tube containing 2 ml of sterilized water. The heat treatment was performed by immersing the tube in a water bath set at each processing temperature for several seconds to several tens of hours. After the heat treatment, the tube was cooled with running water.

(3) Centrifugation treatment The test tissue is immersed in a centrifuge tube containing sterile water, and
Centrifugation was performed at 20,000 G for 1 minute to 60 minutes.

(4) Inoculation and co-cultivation After heat, centrifugation, or a combination treatment, the sterilized water in the tube was removed, a suspension of Agrobacterium was added, and the mixture was stirred with a vortex mixer for 5 to 30 seconds. Preparation of the bacterial suspension was according to Hiei, Y. et al., (13). That is, AB medium (Chilton,
Agrobacterium colonies cultured on MD., Et al., 1974 (Reference (7)) for 3 to 10 days were scraped with a platinum loop and modified AA medium (AA main inorganic salts, AA amino acids and AA vitamins). (Toriyama K. et al., 1985 (Reference (39))), MS trace salts (Murashige, T. et al., 196).
2 (Ref. (32))), 1.0 g / l casamino acid, 100 μM acetosyringone, 0.2 M sucrose, 0.2 M glucose). Also, the bacterial density of the suspension is about 0.3-1 x 10 ⁹ c
Adjusted to fu / ml. After leaving still at room temperature for about 5 minutes, it was placed on a medium for co-culture. The co-culture medium used was 2N6-AS (Hiei et al. 1994) using 8 g / l agarose as a solidifying agent.
(Reference (13))) was used. After co-cultivation in the dark at 25 ° C. for 3 to 7 days, a part of the immature embryos was subjected to a GUS gene expression study by X-Gluc treatment according to the method of Hiei et al. (1994) (13). . That is, immediately after co-culture treatment,
0.1 M phosphate buffer (pH6.8) containing 0.1% Triton X-100
And left at 37 ° C. for 1 hour. After removing Agrobacterium with phosphate buffer, 1.0 mM 5-bromo-
4-chloro-3-indolyl-β-D-glucuronic acid (X
-gluc) and a phosphate buffer containing 20% methanol. After treatment at 37 ° C. for 24 hours, a tissue exhibiting a blue color was observed under a microscope.

(5) Selection of transformed cells (Japonica rice) After co-cultivation, the scutellum site of the immature embryo that had grown and expanded was divided 4-7 using a scalpel, and counted on a 2N6 medium (described above) containing no selection drug. The cells were cultured under light conditions at 30 ° C. for days. Then, 50-100
The cells were transplanted onto a 2N6 medium containing mg / l hygromycin, and cultured at 30 ° C under light conditions for about 2 to 3 weeks. The medium containing 10 mg / l phosphinothricin (PPT) as a selection drug contained 2 mg / l
CC medium (Potrykus e) containing 2.4-D and excluding coconut water
t al. 1979 (Reference (34))) was used. The drug-resistant calli formed on the medium were transplanted to N6-7 medium (Hiei et al. 1994 (reference (13))) containing the same concentration of the selected drug, and incubated for 2 days at 30 ° C under a light condition for 7 days. Next selection was made. Each medium contains
250 mg / l cefotaxime and 250 mg / l carbenicillin disodium were combined, or 250 mg / l cefotaxime alone was added. In addition, 4g / l Gel
rite was used. XG on drug-resistant callus grown on medium
The luc treatment was performed, and the expression of the GUS gene was examined by the above method.

(6) Results Combination of heat and centrifugation and treatment of immature embryos alone and LB
Tables 1 and 2 show the results of transient expression of the GUS gene after co-culture with A4404 (pIG121Hm) and LBA4404 (pNB131). When heat treatment or centrifugation was performed, the GUS expression region in the scutellum was clearly wider and gene transfer occurred more frequently than in the untreated group. Furthermore, the frequency was further increased by combining heat and centrifugation.

The results of selection of transformed calli obtained by co-culturing rice immature embryos and Agrobacterium and then culturing them on a medium containing a selection drug are shown in Tables 3, 4 and 5. . The efficiency of obtaining transformed calli showing drug-resistant and uniform GUS gene expression was
By performing heat or centrifugal treatment, it was significantly improved.
In addition, by combining heat and centrifugation, the transformation efficiency was further improved as compared to each of the single treatments (Table 1).
3, Table 4, Table 5). As described above, it was clarified that the combination of heat and centrifugation treatment on rice immature embryos can be transformed more frequently than each treatment alone.

When the effect of gene transfer by centrifugal treatment alone is low due to differences in varieties, etc., the combined use of heat treatment significantly improves the gene transfer efficiency, and the effect is more frequent than in the heat treatment alone. I also confirmed that. Furthermore, by setting the temperature of the centrifuge at about 40 ° C. during centrifugation, simultaneous processing of centrifugation and heat is possible, and it has been confirmed that the same effect as the above-described combination processing is obtained.

Hiei et al. (1994) (Reference (13))
Report that transformation can be performed with relatively high efficiency using rice calli as a material. Also, Alde
mita RR et al., (Reference (1)) reports a transformation example using rice immature embryos. In order to carry out these transformation techniques more efficiently and stably, the above-mentioned combination treatment method is very effective. In particular, immature embryos are easily affected by the cultivation environment, and it is not easy to always obtain immature embryo material suitable for transformation. However, by performing a combination treatment, stable and high transformation efficiency can be maintained. (1994 (13)) showed that a superbinary vector, which is a vector having high transformation ability, can improve the efficiency of rice transformation.Aldemita RR. Et al., 1996 ( According to the reference (1), transformants were obtained only in the test using the super binary vector LBA4404 (pTOK233) .The combination treatment method in this study was based on the case where a normal binary vector was used. In addition, the gene transfer efficiency can be comparable to or higher than that of the super binary vector, and the efficiency can be further improved by using the combination treatment with the super binary vector. By using the combination treatment method, transformants can be obtained even in varieties where transformants could not be obtained before. It is assumed that possible.

[0042]

[Table 1] Table 1 Transient expression of GUS gene in heat / centrifugation and immature embryo scutellum (variety: Morning light) Test strain: LBA4404 (pIG121Hm), co-culture period: 5 days

[0043]

[Table 2] Table 2 Transient expression of GUS gene in heat / centrifugation and immature embryo scutellum (variety: morning light) Test strain: LBA4404 (pNB131), co-culture period: 6 days

[0044]

[Table 3] Table 3 Heat and centrifugation treatment for immature embryos and selection efficiency of transformed calli (variety: morning light) Test strain: LBA4404 (pIG121Hm), Co-culture period: 5 days, H
m: 100 mg / l hygromycin

[0045]

Table 4 Heat and centrifugation of immature embryos and selection efficiency of transformed calli (variety: morning light) Test strain: LBA4404 (pIG121Hm), Co-culture period: 6 days, H
m: 100 mg / l hygromycin

[0046]

[Table 5] Table 5 Heat and centrifugation of immature embryos and selection efficiency of transformed calli (variety: morning light) Test strain: LBA4404 (pNB131), Co-culture period: 6 days, PP
T: 10 mg / l phosphinothricin

Example 2 An immature maize embryo (variety A188, obtained from the Institute of Bioresources, Ministry of Agriculture, Forestry and Fisheries) of about 1.2 mm in size was aseptically taken out.
Placed in a 2 ml tube containing LS-inf liquid medium. After washing once with the same liquid medium, 2.0 ml of the same liquid medium was newly added. The heat treatment was performed by immersing the tube in a 46 ° C. water bath for 3 minutes. The centrifugation treatment was performed by centrifugation at 20 KG and 4 ° C. for 30 minutes using a cooling centrifuge.
In the combined treatment of heat and centrifugation, the above-mentioned heat treatment was followed by the above-mentioned centrifugal treatment. The control was allowed to stand at room temperature for the same time. After each treatment, remove the medium and remove L containing 100 μM acetosyringone.
At a concentration of about 1 x 10 ⁹ cfu / ml in S-inf liquid medium,
rium tumefaciens LBA4404 (pSB131) (Ishida et al. 19
96 (reference document (20))) was added thereto, and the mixture was stirred with a vortex mixer for 30 seconds. After standing at room temperature for 5 minutes, 10 μM AgNO ₃ was added so that the hypocotyl surface was in contact with the medium.
Was placed on an LS-AS medium containing After culturing in the dark at 25 ° C. for 3 days, some immature embryos were collected and examined for transient expression of the GUS gene by X-gluc. Note that pSB131 is a super binary vector.

The immature embryos after co-culture were cultured in a medium containing phosphinothricin (PPT) and 10 μM AgNO ₃ to select transformed cells. Callus grown on the selection medium
The plant was placed on a regeneration medium containing PPT, and the transformed plant was regenerated. A part of the leaves of the regenerated plant was cut off, and the expression of the GUS gene was examined by X-gluc as in Example 1. The above medium and culture method are described in Ishida, Y. et.
al., 1996 (reference (20)).

LBA4404 (pSB131) was added to the immature embryos after each treatment.
Table 6 shows the results of the transient expression of the GUS gene when inoculated. GUS gene expression was observed in all immature embryos tested, including untreated controls. But,
The expression site was stronger when heat treatment and heat / centrifugation were combined than in the control. In particular, when the combination treatment was performed, the expression of the GUS gene was most frequently observed in a wide portion of the scutellum surface of the immature embryo.

Table 7 shows the results of transformation in immature embryos inoculated with LBA4404 (pSB131). Transformed plants were obtained from control immature embryos that were not heat-treated at an efficiency of 10.7%. On the other hand, the transformation efficiency of the immature embryo subjected to centrifugation at 20 KG at 4 ° C. for 30 minutes was 13.3%, which was higher than that of the untreated embryo. Transformation efficiency in heat-treated immature embryos
At 20%, the efficiency improved about twice as much as no processing. In addition, heat
When combined treatment with centrifugation was performed, the transformation efficiency was 29.6%, which was about three times that of no treatment.

From the above results, the transformation efficiency is improved by centrifuging or heat-treating the immature embryo of the material before inoculation as compared with the conventional method. However, by combining both treatments, the transformation efficiency is further improved. It became clear that it was done. From these facts, transformation of corn varieties other than A188 (Ishida et al. 1996 (reference (20))), which could not be transformed by the conventional Agrobacterium method, by combining heat and centrifugation. The possibility of obtaining plants was suggested.

[0052]

Table 6 Effect of each treatment on gene transfer efficiency (inoculated with LBA4404 (pSB131)) Transient expression of GUS gene in immature embryos after co-culture

[0053]

Table 7 Effect of each treatment on transformation efficiency (LB
A4404 (pSB131) introduced Neither the number of calli nor the number of plants include clones.

Example 3 Creeping bentgrass (Agrostis palustris cv. P
After sterilizing the ripe seeds of Encross, Snow Brand Seed Co., Ltd., MS inorganic salts, MS vitamins, 4 mg / l dicamba, 0.5 mg / l6BA, 0.7
g / l proline, 0.5 g / l MES, 20 g / l sucrose, 3 g / lg
Place on a medium (TG2 medium) containing elrite (pH 5.8),
The cells were cultured at ℃ in the dark. The induced calli were subcultured in a medium having the same composition to grow an embryogenic callus. The resulting embryogenic callus was transferred to a liquid medium (TG2L) having a composition obtained by removing gelrite from TG2, and
Suspension cultured cells were obtained by shaking culture in the dark at ℃. Suspension cultured cells 3-4 days after subculture contain TG2L medium 2
Placed in ml tubes. After washing once with the same liquid medium, 2 ml of a liquid medium was newly added. The tube was immersed in a 46 ° C. water bath for 5 minutes. After removing the medium and adding a fresh liquid medium, the mixture was centrifuged at 5,000 rpm at 4 ° C. for 10 minutes. The control was allowed to stand at room temperature for the same time. Remove medium, 10
About 1 × 10 ^{9 in} TG2-inf medium containing 0 μM acetosyringone (48.46 g / l sucrose, 36.04 g / l glucose added (pH 5.2), excluding proline, MES and gelrite from TG2 medium)
At a concentration of cfu / ml, Agrobacterium tumefaciens LBA4404
1.0 ml of a suspension of (pTOK233) (described above) was added, and the mixture was stirred with a vortex mixer for 30 seconds. After standing at room temperature for 5 minutes, the cells were placed on a medium (TG2-AS medium) in which 10 g / l glucose, 100 μM acetosyringone, and 4 g / l type I agarose (pH 5.8) were added to TG2L medium. 25 ° C, in the dark
After culturing for 3 days, the cells were washed three times with TG2L medium containing 250 mg / l cefotaxime and carbenicillin. The cells were suspended in the same medium and cultured at 25 ° C. in the dark with rotary shaking at 70 rpm. One week later, the same medium was subcultured in a medium containing 50 mg / l hygromycin, and after further culturing for one week, some cells were collected and examined for GUS gene expression by X-gluc.

Table 8 shows the expression of the GUS gene in a cultured suspension of mackerel inoculated with LBA4404 (pTOK233). Only one cell mass of the control cells showed GUS expression. On the other hand, when subjected to heat and centrifugation, about 30% of the cell mass showed GUS gene expression. The GUS gene expression site was larger in the heat- and centrifuged cell mass than in the control cell mass.

Transformation of mackerel so far reported by particle gun (Zhong et al. 1993 (reference (45)),
Hartman et al. 1994 (Ref. (12)), Xiao, L. et a.
l., 1997 (reference (43)), electroporation (Asano Y., 1994 (reference (3)), Asano Y. et al. 199
8 (Reference (4))), and no successful example of Agrobacterium-mediated transformation has been found.
As seen in this example, if the low efficiency of gene transfer by the conventional method is a cause of difficulty in transforming the mackerel by the Agrobacterium method, the present application in which gene transfer is frequently performed is performed. The possibility of obtaining a transformed plant by the combined heat and centrifugation treatment of the present invention was shown.

[0057]

[Table 8] Effect of heat and centrifugation on the efficiency of gene transfer to cultured cells of suspension of mackerel Investigate GUS gene expression 2 weeks after co-culture

[0058]

According to the present invention, gene transfer can be carried out easily and with higher efficiency than the conventional gene transfer method using the Agrobacterium method without damaging the tissue.
A method has been provided for improving the efficiency of gene transfer into plant cells. The method of the present invention is applicable to both monocots and dicots. Also, like Shiva,
Plants that could not be transformed by the conventional Agrobacterium method can now be transformed by the method of the present invention.

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[Brief description of the drawings]

FIG. 1 is a diagram showing a method for constructing pTOK233, which is an example of a super binary vector that can be preferably used in the method of the present invention.

FIG. 2 is a diagram showing a genetic map of pNB131 which is an example of a super binary vector that can be preferably used in the method of the present invention.

FIG. 3 is a schematic diagram showing a process of constructing an intermediate vector system and a binary vector system, which are two main types of vector systems of the genus Agrobacterium.

FIG. 4 is a schematic diagram showing two types of binary vector systems derived from the strongly pathogenic strain A281 of Agrobacterium tumefaciens.

[Explanation of symbols]

BL Left border sequence of T-DNA of Agrobacterium sp. BR Right border sequence of T-DNA of Agrobacterium sp. TC Tetracycline resistance gene SP Spectinomycin resistance gene IG Intron GUS gene HPT Hygromycin resistance gene NPT kanamycin resistance gene K restriction enzyme KpnI site H restriction enzyme HindIII site Amp ^r ampicillin resistance gene bAR bar gene COS, COS site oRI of cos lambda phage, terminator replication P35S CaMV 35S promoter Tnos nopaline synthase gene ori ColE1 virB The virB gene in the virulence region of the Ti plasmid pTiBo542 contained in the Agrobacterium tumefaciens A281 VirG gene in the virulence region of Escherichia coli Vir Total vir of Ti plasmid of Agrobacterium
Region S Vir Whole vir region of Ti plasmid pTiBo542 of strongly pathogenic Agrobacterium s vir * Fragment containing part of vir region of Ti plasmid pTiBo542

Continuation of the front page (72) Inventor Yuji Ishida 700 B. Higashihara, Toyotamachi, Iwata-gun, Shizuoka Japan F-term in the Tobacco Inc. Genetic Breeding Research Institute 4B024 AA08 DA01 EA04 GA11 HA20

Claims (15)

[Claims] 1. A method for improving the efficiency of gene transfer into a plant cell via Agrobacterium, which comprises heat-treating and centrifuging the plant cell or plant tissue. 2. The method according to claim 1, wherein the gene transfer treatment is performed after heat treatment and centrifugation of the plant cell or plant tissue. 3. The method according to claim 1, wherein the heat treatment is performed in a temperature range of 33 ° C. to 60 ° C. 4. The method according to claim 3, wherein the heat treatment is performed in a temperature range of 35 ° C. to 55 ° C. 5. The method according to claim 4, wherein the heat treatment is performed in a temperature range of 37 ° C. to 52 ° C. 6. The method according to claim 1, wherein the heat treatment is performed for a period of from 5 seconds to 24 hours. 7. A temperature of 37 ° C. to 52 ° C. for 1 minute to 24 hours.
3. The method according to claim 1, wherein the heat treatment is performed for a time. 8. The method according to claim 1, wherein the centrifugation is performed at a centrifugal acceleration of 100 G to 250,000 G. 9. The method according to claim 8, wherein the centrifugation is performed at a centrifugal acceleration of 500 G to 200,000. 10. The method according to claim 9, wherein the centrifugation is performed in a centrifugal acceleration range of 1,000 G to 150,000 G. 11. The method according to any one of claims 1 to 10, wherein the centrifugation is performed for 1 second to 4 hours. 12. The method according to claim 1, wherein the plant cell or plant tissue used is derived from an angiosperm. 13. The method according to claim 12, wherein the plant cell or plant tissue used is derived from a monocotyledonous plant. 14. The method according to claim 13, wherein the plant cell or plant tissue used is derived from a gramineous plant. 15. The plant cell or plant tissue used is rice,
15. The method according to claim 14, which is corn or maize.