JP2000342255A - 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, to perform transformation and to better a breed by heat-treating a plant cell or a plant tissue. SOLUTION: A plant cell or a plant tissue derived from a plant selected from the group consisting of rice plant, maize, lawn grass and tobacco 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, especially preferably at 37-52 deg.C for 5 minutes to 24 hours to improve the efficiency of gene transfer to a plant cell carried out through a bacterium of the genus Agrobacterium. Preferably the plant cell or plant tissue is derived from an angiosperm, a monocotyledon or a gramineous plant. Preferably after the plant cell or plant tissue is heat-treated or centrifuged, 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 (35)). 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 the plant tissue used as a material without sterilization as necessary, but without any special treatment (Rogers et al. 1988 (Ref. (36) ), Visser 199
1 (Reference (40)), 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).
(Ref. (6)) and ultrasound (Trick et al., 1997 (Ref. (39))). 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 not the case with the more widely used leaf-disc method (Horsch et al., 1985 (ref.
It is just an extension of (19))) 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 intensive studies, the present inventors have conducted a gene transfer method using a bacterium belonging to the genus Agrobacterium by heat-treating a plant cell or a plant tissue to be subjected to gene transfer. The present inventors have found that the efficiency can be significantly improved and completed the present invention.

[0008] That is, the present invention provides a method for improving the efficiency of gene transfer into a plant cell via Agrobacterium, which involves heat-treating the plant cell or plant tissue.

[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-treating a plant cell or a plant tissue into which the gene is to be introduced. The plant cells or plant tissues may be heat treated and cooled to room temperature and then contacted with Agrobacterium bacteria, or may be heat treated and contacted with Agrobacterium bacteria. Alternatively, a method may be used in which after heat treatment and cooling to room temperature, the mixture is brought into contact with Agrobacterium bacteria while heat treatment. Among these methods, a preferable method is a method of performing heat treatment, cooling to room temperature, and then contacting with a Agrobacterium bacterium.

The heat treatment conditions are appropriately selected according to the type of plant to be used, the amount of cells or tissues to be heat-treated, etc., but are usually 33 ° C to 60 ° C, preferably 35 ° 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 method of the present invention is characterized in that heat-treated plant cells or plant tissues to be brought into contact with Agrobacterium bacteria are used, or the plant cells or plant tissues are brought into contact with Agrobacterium bacteria while performing the heat treatment. ,
As a method for gene introduction or transformation using Agrobacterium bacteria, well-known methods can be directly applied.

[0012] The method of 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 on many dicotyledonous plants in apical 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).

T-DNA by Agrobacterium infection
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 (references (14)), C58C1 (pGV3850) (Zambryski et
al., 1983 (Reference (43))), GV3Ti11SE (Fraley et a
l., 1985 (Reference (10)) and the like were produced (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. This method is introduced by homologous recombination via a triparental mating method (Ditta et al., 1980 (reference (9))) and is called an intermediate vector method (Fraley et al., 1985).
(Reference (10)); Fraley et al., 1983 (Reference (1)
1)); Zambryski et al., 1983 (Reference (43)), JP-A-59-140885 (EP116718)). The other is called the binary vector method (Fig. 3), which requires a vir region for integration of T-DNA into plants, but it must be on the same plasmid to function. Result (Hoekema et al., 1983 (references (14))
Based on This vir area contains virA, virB, virC, v
irD, virE and virG exist, (Encyclopedia of Plant Biotechnology (published by Enterprises, Inc. (1989))), vi
r region is virA, virB, virC, virD, virE and virG
Means all of the above. Therefore, a binary vector is one in which T-DNA is integrated into a small plasmid that is replicable in both Agrobacterium and E. 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 (references).
(5))), pBI121 (Jefferson, 1987 (Reference (21))), pGA4
82 (An et al., 1988 (Reference (2))), JP-A-60-70080
(EP120516)), and 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 (Ref. (41)) is strongly virulent (super-virulen
t), its host range is wide, and the transformation efficiency is higher than other strains (Hood et al., 1987 (Ref. (15));
Komari, 1989 (Ref. (23)). This property is due to the Ti plasmid pTiBo542 carried by A281 (Hood et al.
al., 1984 (ref. (18)); Jin et al., 1987 (ref. (22)); Komari et al., 1986 (ref. (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
(Reference (25)); Li et al. (Reference (29)), 1996; Sai
to et al., 1992 (reference (37)).

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 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 through Agrobacterium can be performed by simply contacting a plant cell or a 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).

[0026]

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 Bacteria Morning light of Japonica rice and IR72 of indica rice were used as test varieties, and immature embryos and calli derived from 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 et al., 1994 (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
It was obtained by culturing on a medium (Hiei et al. 1994 (reference (13))) 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
B31 (Ishida et al., 1996 (reference (20)) was transformed into E. coli LE392.
After introduction into the strain, the parental mating method (Ditta et al.,
1980 (reference (9)), pNB1 (Komari et al., 19
Agrobacterium LBA4404 with 96 (reference (27))
Introduced into the strain. PNB131 was obtained by homologous recombination between pNB1 and pSB31 in Agrobacterium.

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. GUS gene controlled by 35S promoter and mediated by intron of castor catalase gene (Ohta et al, 1990 (Reference (33)))
Having.

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 et al., 1999 (reference (28))).

(2) Heat treatment The test tissue (5-200 mg) was immersed in a tube containing 1 ml of sterilized water. The heat treatment was performed by immersing the tube in a water bath set at each processing temperature for 1 minute to several tens of hours. Control treatments were allowed to stand at room temperature (25 ° C.). After the heat treatment, the tube was cooled with running water.

(3) Inoculation and co-cultivation After heat treatment, sterile water in the tube was removed, an Agrobacterium suspension was added, and the mixture was stirred with a vortex mixer for 5 to 30 seconds. Preparation of bacterial suspensions is described in Hiei et.
al. (1994) ((13).
Agrobacterium colonies cultured on B medium (Chilton M-D et al., 1974 (reference (8)) 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, 1985
(Reference (38))), MS trace salts (Murashige T., 1962)
(Reference (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 cfu
/ ml. After leaving still at room temperature for about 5 minutes, it was placed on a medium for co-culture. The medium for co-culture of immature embryos contains 8 g /
l 2N6-AS using agarose as a medium solidifying agent (Hiei et al.
1994 (Reference (13))). The medium for callus coculture was 2N6-AS containing 4 g / l Gelrite (Hiei et al. 1994).
(Reference (13))) was used. After co-culturing at 25 ° C. in the dark for 3 to 7 days, a part of the immature embryo or callus was examined for the expression of the GUS gene by X-Gluc treatment according to the method of Hiei et al. (1994) (reference (13)). Provided. That is, immediately after the co-culture treatment, the tissue was immersed in 0.1 M phosphate buffer (pH 6.8) containing 0.1% Triton X-100, and allowed to stand at 37 ° C for 1 hour.
After removing Agrobacterium with phosphate buffer,
mM 5-bromo-4-chloro-3-indolyl-β-D
-A phosphate buffer containing glucuronic acid (X-gluc) and 20% methanol was added. After treatment at 37 ° C. for 24 hours,
The tissue exhibiting a blue coloration was observed under a microscope.

(4) Selection of Transformed Cells in Japanese Rice After co-culture, immature embryos were divided into 4 to 7 scutellum scutellum sites with a scalpel for calligenic embryos. 3 days in 2N6 medium without medium (described above)
The cells were cultured under light conditions at 0 ° C. Next, 2N containing either 50-100 mg / l hygromycin, 200 mg / l paromomycin
The cells were transplanted onto a 6-medium and cultured at 30 ° C under light for about 2 to 3 weeks. The medium containing 10 mg / l phosphinothricin (PPT) as a selection drug includes a CC medium containing 2 mg / l 2.4-D and excluding coconut water (Potrykus et al. 1979 (Reference (3)
4))) was used. The drug-resistant callus formed on the medium was transferred to an N6-7 medium (Hiei e
t al. 1994 (Reference (13)), and a second selection was carried out at 30 ° C. under light conditions for 7 days. Each medium was combined with 250 mg / l cefotaxime and 250 mg / l carbenicillin disodium or added 250 mg / l cefotaxime alone. In addition, 4 g / l Gelrite was used as a medium solidifying agent. X-Gluc treatment was applied to the drug-resistant calli grown on the medium, and GUS
The expression of the gene was investigated.

(5) Results Immature embryos were treated at various treatment temperatures for 10 minutes, and LBA4404 (pTOK23
Table 1 and Table 2 show the results of investigating the transient expression of the GUS gene by co-culture with 3). 46 compared to untreated area
It was understood that the GUS expression region in the scutellum was clearly wider when the heat treatment was performed at around ℃, and gene transfer occurred more frequently. On the other hand, heat treatment at 52 ° C or higher could damage plant tissues, and treatment at 55 ° C suppressed the growth of immature embryos, and no GUS expression was observed. In the short-time treatment for 10 minutes, no clear expansion of the GUS expression region in the 43 ° C test group was observed.

Table 3 and Table 4 show 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. LB
In a test inoculated with A4404 (pIG121Hm), transformed calli showing hygromycin-resistant and uniform expression of the GUS gene were obtained at an efficiency of 12.0% from split sections of immature embryos that had not been heat-treated (Table 4). ). On the other hand, transformed calli were obtained with a 29% efficiency in divided sections of immature embryos that had been heat-treated at 46 ° C for 5 minutes (Table 3). In addition, in the selection with paromomycin, transformed calli were obtained at a low efficiency of 2.0% from the section of the immature embryo that had not been heat-treated, while the immature embryo section that had been heat-treated at 46 ° C for 5 minutes had Obtained with an efficiency of 12.0% (Table 4). LBA4404 (pNB13
In a test inoculated with 1), a transgenic callus showing PPT resistance and uniform expression of the GUS gene was obtained at a 29.0% efficiency from a section of the immature embryo that had not been heat-treated (Table 5). In contrast, in the section of the immature embryo that had been heat-treated at 46 ° C. for 5 minutes, transformed calli were obtained with an efficiency of 50.0% (Table 5). In all cases, heat treatment of immature embryos significantly improved the transformation efficiency.

[0038] Table 6 shows the results of selection of transformed calli obtained by co-culturing rice calli derived from immature embryos and Agrobacterium LBA4404 (pTOK233) and then culturing on a medium containing hygromycin. Non-heat treated calli yield a hygromycin resistant and uniform GU with 24.1% efficiency.
Transformed calli showing S gene expression were obtained (Table 6). On the other hand, transformed calli were obtained with an efficiency of 36.2 to 36.9% in the callus that was heat-treated at 46 ° C. for 3 minutes or 10 minutes (Table 6). As described above, also in the heat treatment of rice calli, a remarkable improvement in transformation efficiency was observed.

Using immature embryos and calli, the relationship between the treatment temperature and the treatment time was further examined using GUS expression after co-culture as an index. In the case of temperature, long-term processing,
Increased efficiency of gene transfer. That is, a clear effect of improving the gene transfer efficiency was observed by the treatment for several seconds in the heat treatment at 60 ° C. and by the treatment for several hours to several tens of hours at 35 ° C.

Hiei et al. (1994) (reference (13))
It reports that transformation can be performed with relatively high efficiency using rice calli as a material. Also, Aldemita
RR. Et al., 1996 (Reference (1)) reports a transformation example using a rice immature embryo. In order to carry out these transformation techniques more efficiently and stably, the above-mentioned heat 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, but heat treatment can maintain stable and high transformation efficiency. Hiei e
t al. (1994) (ref. (13)) showed that a super binary vector, which is a vector having high transformation ability, improves the transformation efficiency of rice. Also Aldemi
According to ta and Hodges (1996) (Reference (1)), transformants were obtained only in tests using the super binary vector LBA4404 (pTOK233). The heat treatment method in this study can achieve gene transfer efficiency comparable to or higher than that of a super binary vector even when a normal binary vector is used. In addition, the efficiency can be further improved by using the super binary vector and the heat treatment method together. Furthermore, it is presumed that a transformant can be obtained even in a variety in which a transformant could not be obtained at all by using the heat treatment method.

[0041]

Table 1. Transient expression of GUS gene in the treatment temperature and immature embryo scutellum (variety: IR72) Test strain: LBA4404 (pTOK233), heat treatment time: 10 minutes, co-culture period: 4 days

[0042]

Table 2 Transient expression of GUS gene in treatment temperature and immature embryo scutellum (variety: morning light) Test bacterial system: LBA4404 (pTOK233), heat treatment time: 10 minutes, co-culture period: 3 days

[0043]

[Table 3] Table 3 Heat treatment of immature embryos and selection efficiency of transformed calli (variety: morning light) Test bacterial system: LBA4404 (pIG121Hm), Co-culture period: 5 days, H
m: Hygromycin

[0044]

[Table 4] Table 4 Heat treatment of immature embryos and selection efficiency of transformed calli (variety: morning light) Test strain: LBA4404 (pIG121Hm), Co-culture period: 5 days, P
m: Paromomycin

[0045]

[Table 5] Heat treatment of immature embryos and selection efficiency of transformed calli (variety: morning light) Test strain: LBA4404 (pNB131), co-culture period: 6 days

[0046]

[Table 6] Table 6 Heat treatment of calli and selection efficiency of transformed calli (variety: morning light) Test strain: LBA4404 (pTOK233), Co-culture period: 3 days

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 tube was immersed in a 46 ° C. water bath for 1-10 minutes. The control was allowed to stand at room temperature for the same time. Remove medium, 10
About 1 x in LS-inf liquid medium containing 0 mM acetosyringone
At a concentration of 10 ⁹ cfu / ml, Agrobacterium tumefaciens LBA4
404 (pIG121Hm), EHA101 (pIG121Hm), LBA4404 (pTOK233)
(The above are described in Hiei et al., 1994 (reference (13))), LB
1.0 ml of a suspension of A4404 (pSB131) (Ishida et al., 1996 (Reference (20))) or LBA4404 (pNB131) (described in Example 1 and FIG. 2) was added, and the mixture was stirred with a vortex mixer for 30 seconds. did. After standing at room temperature for 5 minutes, the cells were placed on an LS-AS medium containing 10 μM AgNO ₃ so that the hypocotyl surface was in contact with the medium. 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. The immature embryos inoculated with LBA4404 (pSB131) and LBA4404 (pNB131) were co-cultured and then cultured in a medium containing phosphinothricin (PPT) and 10 μM AgNO ₃ to select transformed cells. The calli grown on the selection medium were placed on a regeneration medium containing PPT to regenerate the transformed plants. A part of the leaves of the regenerated plant was cut off, and the expression of the GUS gene was examined by X-gluc in the same manner as in Example 1. In addition, the above-mentioned medium and culture method
The method described in hida et al. 1996 (reference (20)) was followed. pSB131 and pTOK233 are super binary vectors with high transformation ability.

LBA4404 was added to immature embryos treated at 46 ° C. for 110 minutes.
Table 7 shows the results of transient expression of the GUS gene when (pSB131) was inoculated. GUS gene expression was observed in all immature embryos tested, including untreated controls.
However, the expression site was stronger when heat-treated than the control. In particular, when heat treatment was performed for 3 minutes or more, many of the immature embryos showed GUS gene expression in a wide part of the scutellum surface. Table 8 shows the results of transient expression of the GUS gene when inoculated with various strains of Agrobacterium. In the heat-treated immature embryos, all immature embryos showed GUS gene expression regardless of the inoculation of any strain. In contrast, untreated immature embryos showed weaker expression than any heat-treated strain. In particular, when immature embryos were inoculated with LBA4404 (pIG121Hm) and LBA4404 (pNB131), which are ordinary binary vectors having no virulent gene, most immature embryos did not show GUS gene expression at all.

The super binary vector LBA440
Table 9 shows the results of transformation of A188 immature embryos inoculated with 4 (pSB131). 10.7 from control immature embryos without heat treatment
Transformed plants were obtained with an efficiency of%. In contrast, 46
The transformation efficiency of the immature embryos that had been heat-treated at ℃ for 3 minutes was 20%, which was about twice that of the untreated embryos. LBA4, a regular binary vector without a virulent gene
Table 4 shows the results of the transformation of A188 immature embryos inoculated with 404 (pNB131). No transgenic plants were obtained from control immature embryos that had not been heat treated. In contrast, two independent transformed plants were obtained from the immature embryos that had been heat-treated at 46 ° C for 5 minutes. Transformation efficiency was 2.4%.

From the above results, when the superbinary vector is used, heat transfer of the immature embryo of the material before inoculation can increase the gene transfer efficiency and the transformation efficiency as compared with the conventional method. It became clear. Furthermore, a transformed plant was obtained for the first time by heat treatment even in a conventional binary vector, which has not been successful in corn. From these facts, corn varieties other than A188 which could not be transformed by the conventional Agrobacterium method (Ishida et al. 1996 (references)
(20))), it was suggested that transgenic plants could be obtained by heat treatment.

[0051]

Table 7 Effect of heat treatment time on gene transfer efficiency (inoculated with LBA4404 (pSB131)) Transient expression of GUS gene in immature embryos after co-culture

[0052]

Table 8 Effect of heat treatment on gene transfer efficiency Transient expression of GUS gene in immature embryos after co-culture

[0053]

Table 9 Effect of heat treatment on transformation efficiency (LB
A4404 (pSB131) introduced Neither the number of calli nor the number of plants include clones.

[0054]

[Table 10] Effect of heat treatment on transformation efficiency (LBA4404 (pNB131) was introduced) Neither the number of calli nor the number of plants include clones. Transient expression was investigated by collecting some calli after co-culture.

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 / l
Place on a medium (TG2 medium) containing gelrite (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. After passage, about 0.3 g of callus at 23 weeks was placed in a 2 ml tube containing TG2L medium. 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. The control was allowed to stand at room temperature for the same time. The medium was removed, and TG2-inf medium containing 100 μM acetosyringone (proline, MES, gel
Except rite, 48.46 g / l sucrose, at a concentration of 36.04 g / l glucose was added (pH 5.2)) to about ^{1 x 10 9 cfu / ml,} LBA440
1.0 ml of a suspension of 4 (pTOK233) (Hiei et al., 1994 (reference (13))) was added, and the mixture was stirred with a vortex mixer for 30 seconds. After standing at room temperature for 5 minutes, add 10 minutes to TG2L medium.
Medium (TG2-AS medium) supplemented with g / l glucose, 100 μM acetosyringone, and 4 g / l type I agarose (pH 5.8)
Was placed on the floor. After culturing at 25 ° C. in the dark for 3 days, a portion of the callus was collected, and GU was subjected to GU by X-gluc in the same manner as in Example 1.
The transient expression of the S gene was investigated.

GU in callus inoculated with LBA4404 (pTOK233)
Table 11 shows the transient expression of the S gene. Control callus without heat treatment showed no expression, whereas heat treatment showed transient expression of the GUS gene in 25% of the calli.

Transformation of mackerel, which has been reported so far, is a particle gun (Zhong et al. 1993 (reference (44)),
Hartman et al. 1994 (Ref. (12)), Xiao and Ha 19
97 (references (42))) and electroporation (Asan
o and Ugaki 1994 (Reference (3)), Asano et al. 1998
(Reference (4))), but no successful example of Agrobacterium-mediated transformation was 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 heat treatment of the present invention showed the possibility of obtaining a transformed plant.

[0058]

[Table 11] Table 11 Results of Gene Transfer into Shiva Callus (LBA4
404 (pTOK233) inoculated)

Example 4 (1) Test tissue and test bacterial system Suspension cultured cell line BY2 of tobacco variety Bright Yellow 2
Was used as a test material. 0.2mg / l 2,4- suspended cells
The cells were cultured in an LS liquid medium containing D under dark conditions at 25 ° C., and maintained by subculture every week. Agrobacterium and its vectors include LBA4404 (pTOK233) (Hiei et a
l., 1994 (Reference (13))).

(2) Heat treatment On the fourth day after the passage, about 0.3 g of the suspension cultured cells was immersed in a tube containing 1 ml of sterilized water. Tube at 43 ° C or 46 ° C
Heat treatment was performed by immersion in a water bath set for 10 to 20 minutes. Control treatments were allowed to stand at room temperature (25 ° C.). After the heat treatment, the tube was cooled with running water.

(3) Inoculation and co-cultivation Agrobacterium was inoculated and co-cultured into the suspension culture cells according to the method of Komari (1989) (Reference (23)). After co-cultivation in the dark at 25 ° C. for 2 days, the suspension cultured cells were subjected to the X-Gluc treatment of the GUS gene in the same manner as in Example 1 by the method of Hiei et al. (1994) (Reference (13)). It was subjected to expression studies.

(4) Results The suspension culture cells were heat-treated and co-cultured with LBA4404 (pTOK233), and the results of investigating the transient expression of the GUS gene were shown in Table 12. When heat treatment is performed compared to the untreated area, GU
The frequency of cells showing S expression was clearly higher, indicating that gene transfer occurred more frequently. It was confirmed that the heat treatment had an effect of improving the efficiency of gene transfer not only to monocotyledonous plants such as rice, maize and maize, but also to dicotyledonous plants.

[0063]

[Table 12] Treatment temperature and transient expression of GUS gene in suspension culture cell BY2 * +: Low, ++: slightly high, +++: high Test strain: LBA4404 (pTOK233), co-cultivation period: 2 days

[0064]

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.

References (1) Aldemita RR, Hodges TK (1996) Agrobacterium tu
mefaciens-mediated transformation of japonica and
indica rice varieties.Planta 199: 612-617 (2) An, G., Evert, PR, Mitra, A. and Ha, SB (1
988) Binary vectors.In Gelvin, SB and Schilpero
ort, RA (eds.), Plant Molecular Biology Manual A
3. Kluwer Academic Press, Dordrecht, pp. 1-19. (3) Asano, Y., Ugaki, M. (1994) Transgenic plants
of Agrostis alba obtained by electroporation-media
ted direct gene transfer into protoplasts.Plant C
ell Reports 13: 243-246. (4) Asano, Y., Ito, Y., Fukami, M., Sugiura, K., F
ujiie, A. (1998) Herbicide-resistant transgenic cr
eeping bentgrass plants obtained by electroporatio
n using an altered buffer.Plant Cell Reports 17: 9
63-967. (5) Bevan, M. (1984) Binary Agrobacterium vectors
for plant transformation.Nucleic Acids Res., 12,
8711-8721. (6) Bidney, D., Scelonge, C., Martich, J., Burrus,
M., Sims, L., and Huffmanm G. (1992) Microproject
ile bombardment of plant tissues increases transfo
rmation frequency by Agrobacterium tumefaciens. Pl
ant Mol. Biol., 18, 301-313 (7) Chan, MT., Cheng, HH., Ho, SL., Tong, WF.
and Yu, SM. (1993) Agrobacterium-mediated product
ion of transgenic rice plants expressing a chimeri
c α-amylaze promoter / β-glucuronidase gene. (8) Chilton, MD., Currier, TC., Farrand, SK., Ben
dich, AJ., Gordonm MP. & Nester, EW. (1974) Agrobac
terium tumefaciens DNA and PS8 bacteriophage DNA n
ot detected in crown gall tumers. Proc. Natl. Aca
d. Sci. USA, 71: 3672-3676. (9) Ditta, G., Stanfield, S., Corbin, D. and Helin.
ski, DR (1980) Broadhost range DNA cloning syste
m for Gram-negative bacteria: Constructionof gene
Bank of Rhizobium meliloti. Proc. Natl. Acad. Sci.
USA, 77, 7347-7351. (10) Fraley, RT, Rogers, SG, Horsch, RB, Eic
holtz, DA and Flick, JS (1985) The SEV system:
a new disarmed Ti plasmid vector for planttransfor
mation. Bio / technology, 3, 629-635. (11) Fraley, RT, Rogers, SG, Horsch, RB, San.
ders, PR, Flick, JS, Adams, SP, Bittner, M.
L., Brand, LA, Fink, CL, Fry, JS, Galluppi,
GR, Goldberg, SB, Hoffmann, NL and Woo, SC
(1983) Expression of bacterial genes in plant cel
ls. Proc Natl Acad Sci USA, 80, 4803-4807. (12) Hartman, CL, Lee, L., Day, PR, Tumer, N.
E. (1994) Herbicide resistant turfgrass (Agrostis
palustris Huds.) by biolistic transformation.Biote
chnology 12: 919-923. (13) Hiei, Y., Ohta, S., Komari, T. and Kumashiro,
T. (1994) Efficient transformation of rice (Oryza
sativa L.) mediated by Agrobacterium and sequence
analysis of the boundaries of the T-DNA. The Plan
t Journal, 6, 271-282. (14) Hoekema, A., Hirsch, PR, Hooykaas, PJJ a
nd Schilperoort, RA (1983) A binary plant vector
strategy based on separation of vir- and T-region
of the Agrobacterium tumefaciens Ti-plasmid. Natur
e, 303, 179-180. (15) Hood, EE, Fraley, RT and Chilton, M.-D.
(1987) Virulence of Agrobacterium tumefaciens stra
in A281 on legumes.Plant Physiol, 83, 529-534. (16) Hood, EE, Gelvin, SB, Melchers, LS and
Hoekema, A. (1993) NewAgrobacterium helper plasmid
s for gene transfer to plants.Transgenic Res., 2,
208-218. (17) Hood, EE, Helmer, GL, Fraley, RT and Ch
ilton, M.-D. (1986) The hypervirulence of Agrobact
erium tumefaciens A281 is encoded in a region of p
TiBo542 outside of T-DNA. J. Bacteriol., 168, 1291
-1301. (18) Hood, EE, Jen, G., Kayes, L., Kramer, J., F
raley, RT and Chilton, M.-D. (1984) Restriction
endonuclease map of pTiBo542, a potential Ti-plasm
id vector for genetic engineering of plants.Bio/t
echnology, 2, 702-709. (19) Horsch, RB, Fry, JE, Hoffmann, NL, E
ichholtz, D., Rpgers, SG and Fraley, RT (1985)
A simple and general method for transferring gene
s into plants.Science 227, 1229-1231. (20) Ishida, Y., Saito, H., Ohta, S., Hiei, Y., Ko
mari, T. and Kumashiro, T. (1996) High efficiency
transformation of maize (Zea mays L.) mediated by
Agrobacterium tumefaciens.Nature Biotechnol, 14,
745-750. (21) Jefferson, RA (1987) Assaying chimeric gene
s in plants: the GUS gene fusion system.Plant Mo
l. Biol. Rep., 5, 387-405. (22) Jin, S., Komari, T., Gordon, MP and Nester,
EW (1987) Genes responsible for the supervirule
nce phenotype of Agrobacterium tumefaciens A281.
J. Bacteriol., 169, 4417-4425. (23) Komari, T. (1989) Transformation of callus cu
ltures of nine plant species mediated by Agrobacte
rium. Plant Sci., 60, 223-229. (24) Komari, T. (1990a) Genetic characterization o
f double-flowered tobacco plant obtained in a tran
sformation experiment. Theor. Appl. Genet., 80, 167
-171. (25) Komari, T. (1990b) Transformation of cultured
cells of Chenopodiumquinoa by binary vectors that
carry a fragment of DNA from the virulenceregion
of pTiBo542.Plant Cell Reports, 9, 303-306. (26) Komari, T., Halperin, W. and Nester, EW (19
86) Physical and functional map of supervirulent A
grobacterium tumefaciens tumor-inducing plasmid pT
iBo542. J. Bacteriol., 166, 88-94. (27) Komari, T., Hiei, Y., Saito, Y., Murai, N. an
d Kumashiro, T. (1996) Vectors carrying two separat
e T-DNAs for co-transformation of higher plants me
diated by Agrobacterium tumefaciens and segregatio
n of transformants free from selection markers.Pl
ant J, 10, 165-174. (28) Komari, T. and Kubo, T. (1999) Methods of Gen
etic Transformation: Agrobacterium tumefaciens. In
Vasil, IK (ed.) Molecular improvement ofcereal
crops. Kluwer Academic Publishers, Dordrecht, pp.
43-82. (29) Li, H.-Q., Sautter, C., Potrykus, I. and Puon
ti-Kaerlas, J. (1996) Genetic transformation of cas
sava (Manihot esculenta Crantz). Nature Biotechno
l., 14, 736-740. (30) Lindsey, K., Gallois, P. and Eady, C. (1991)
Regeneration and transformation of sugarbeet by Ag
robacterium tumefaciens.Plant Tissue Culture Manu
al B7: 1-13.Kluwer Academic Publishers. (31) McCormick, S. (1991) Transformation of tomato
with Agrobacterium tumefaciens.Plant Tissue Cult
ure Manual B6: 1-9. Kluwer Academic Publishers. (32) Murashige, T. and Skoog, F. (1962) Physiol. P
lant 15: 473-497. (33) Ohta, S., Mita, S., Hattori, T., Nakamura,
K., (1990) Construction and expression in tobacco o
f aβ-glucuronidase (GUS) reporter gene containing
an intron within the coding sequence.Plant Cell
Physiol. 31: 805-813. (34) Potrykus I., Harms, CT and Lorz, H. (1979)
Callus formation from cell culture protoplasts of
corn (Zea mays L.). Theor. Appl. Genet. 54: 209-21
4. (35) Potrykus, I., Bilang, R., Futterer, J., Sautt
er, C. and Schrott, M. (1998) Agricultural Biotecno
logy, NY: Mercel Dekker Inc. pp. 119-159. (36) Rogers, SG, Horsch, RB and Fraley, RT
(1988) Gene transfer in plants: Production of tran
sformed plants using Ti plasmid vectors.Method fo
r Plant Molecular Biology, CA: Academic Press Inc.
pp.423-436. (37) Saito, Y., Komari, T., Masuta, C., Hayashi,
Y., Kumashiro, T. and Takanami, Y. (1992) Cucumber
mosaic virus-tolerant transgenic tomato plants ex
Pressing a satellite RNA. Theor. Appl. Genet., 83,
679-683. (38) Toriyama, K. and Hinata, K. (1985) Plant Sc
i., 41: 179-183. (39) Trick, HN and Finer, JJ (1997) SAAT: soni
cation-assisted Agrobacterium-mediated transformat
ion.Transgenic Research 6: 329-336. (40) Visser, RGF (1991) Regeneration and transf
ormation of potato byAgrobacterium tumefaciens.Pl
ant Tissue Culture Manual B5: 1-9.Kluwer Academic
Publishers. (41) Watson, B., Currier, TC, Gordon, MP, Chil
ton, M.-D. and Nester, EW (1975) Plasmid required
for virulence of Agrobacterium tumefaciens.J Bac
teriol, 123, 255-264. (42) Xiao, L., Ha, S.-B. (1997) Efficient selectio
n and regeneration ofcreeping bentgrass transforma
nts following particle bombardment.Plant Cell rep
orts 16: 874-878. (43) Zambryski, P., Joos, H., Genetello, C., Leema
ns, J., Van Montagu, M. and Schell, J. (1983) Tip
lasmid vector for the introduction of DNA into pla
nt cells without alteration of their normal regene
ration capacity.EMBO J, 2, 2143-2150. (44) Zhong, H., Bolyard, MG, Srinivasan, C., Sti
cklen, MB (1993) Transgenic plants of turfgrass
(Agrostis palustris Huds.) From microprojectile bo
mbardment of embryogenic callus.Plant Cell Report
s 13: 1-6.

[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 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

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Yuji Ishida 700 Higashihara, Toyota-cho, Iwata-gun, Shizuoka Japan F-term in the Tobacco Inc. Genetics Breeding Research Institute 2B030 AA02 AB03 AD20 CA19 CB03 CD03 CD06 CD09 CD13 CD14 CD17 4B024 AA08 DA01 GA11 HA20

Claims (11)

[Claims] 1. A method for improving the efficiency of gene transfer into a plant cell via Agrobacterium, which comprises heat-treating the plant cell or plant tissue. 2. After heat-treating a plant cell or plant tissue,
The method according to claim 1, wherein the gene transfer treatment is performed. 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 plant cell or plant tissue used is derived from an angiosperm. 9. The method according to claim 8, wherein the plant cells or plant tissues used are derived from monocotyledonous plants. 10. The method according to claim 9, wherein the plant cell or plant tissue used is derived from a gramineous plant. 11. The plant cell or plant tissue to be used is rice,
The method according to claim 8, wherein the method is derived from a plant selected from the group consisting of corn, turf, and tobacco.