JP2001029075A - Extremely rapid transformation of monocoty ledon - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for more efficiently and more rapidly transforming a monocot such as Oryza sativa, Triticum sativum, Hordeum vulgare, and Zea mays by infecting an intact germinating seed with an agrobacterium containing a desired recombinant gene. SOLUTION: This is a method for more efficiently and more rapidly transforming a monocot such as Orya sative by removing a hull from a seed of the monocot, sterilizing the resultant intact seed in a solution such as 2.5% sodium hyposhlorite solution, adequately washing the sterilized seed with water, inoculating the washed seed onto a medium, incubating the inoculated seed at 27-32 deg.C for 4-5 days for germination, soaking the incubated seed in a suspension of a transformed agrobacterium containing a desired recombinant gene to infect the seed with the transformed agrobacterium, transplanting the infected seed onto a medium, incubating the transplanted seed in the dark at 28 deg.C for 3 days for co-culture, followed by washing the agrobacterium from the seed with a medium containing a herbicide such as carbenicillin after the co-culture.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for Agrobacterium-mediated transformation of monocotyledonous plants.

[0002]

2. Description of the Related Art One of means for improving a plant is a "transformation method", in which a desired recombinant gene for modifying a trait is introduced into a plant. Efficient and rapid transformation methods are crucial in the molecular breeding of useful plants, especially cereals, which are important foodstuffs as staple foods.

[0003] Many of the cereals (eg, rice, wheat, barley, and corn) are classified as monocots. Various transformation methods have been developed to transform monocotyledonous plants. Transformation methods are broadly classified into direct transformation methods and indirect transformation methods.

As a direct transformation method, for example, an electroporation method (Shimamoto K. et al.) Is used.
Nature, 338: 274-276, 198.
9; and Rhodes C.I. A. Et al., Sciencec
e, 240: 204-207, 1989), particle gun method (Christou P. et al., Bio /
Technology 9: 957-962, 1991.
And the polyethylene glycol (PEG) method (Data, SK et al., Bio / Technolo).
gy, 8: 736-740, 1990). The electroporation method and the particle gun method have been generally used for transforming monocotyledonous plants as a method capable of relatively efficiently introducing a gene.

[0005] Indirect transformation methods include Agrobacterium-mediated transformation methods (hereinafter sometimes referred to as Agrobacterium transformation methods). Agrobacterium is a kind of plant pathogenic bacteria. When Agrobacterium infects a plant, its own plasmid (eg, Ti plasmid or Ri plasmid)
It has the property of incorporating the upper T-DNA region into plants. In the Agrobacterium transformation method, integration of this T-DNA region into a plant is used as a means for introducing a gene into a plant. Briefly, plants are infected with Agrobacterium containing the desired recombinant gene. After infection, the desired recombinant gene is transferred from Agrobacterium into plant cells and integrated into the plant genome.

[0006] The Agrobacterium transformation method is well established for dicotyledonous plants and to date,
Many stable transformed plants expressing the desired recombinant gene have been produced.

[0007] In contrast, applying Agrobacterium transformation methods to monocotyledonous plants has hitherto generally been difficult. For example, Portrykus et al. (BI
O / TECHNOLOGY, 535-542,199
0) report that Agrobacterium does not infect monocotyledonous plants. However, on the other hand, many attempts have been made to transform monocotyledonous plants using Agrobacterium, and as a result, the possibility of applying the Agrobacterium transformation method to monocotyledonous plants has been found.

For example, Raineri et al. Removed the scutellum of a rice plant, wounded it, placed it on a medium for inducing dedifferentiation, and several days later infected the scutellum with Agrobacterium. As a result, it was not possible to obtain a normal redifferentiated individual, but succeeded in inducing calli into which a foreign gene had been introduced (Raineri, D. et al.
M. Et al., Bio / Technology, 8: 33-3.
8, 1990).

WO 94/00977 discloses an Agrobacterium transformation method for rice and corn. In this method, as a plant sample for transformation with Agrobacterium,
It requires the use of cultured tissue that is in the process of dedifferentiation or has been dedifferentiated (eg, callus). For this reason,
Prior to infection with Agrobacterium, a dedifferentiation induction period of 3 to 4 weeks is usually required to produce a dedifferentiated culture from a plant sample to be transformed (eg, leaf section). I do.

WO 95/06722 discloses a method of infecting immature embryos of rice and corn with Agrobacterium. However, the operation for removing the immature embryo requires a great deal of labor.

Therefore, if a more rapid and efficient method for Agrobacterium transformation of monocotyledonous plants is available,
It can greatly contribute to molecular breeding of useful monocotyledonous plants including grains such as rice.

[0012]

SUMMARY OF THE INVENTION The present invention is intended to solve the above-mentioned problems. An object of the present invention is to provide an improvement in a method for Agrobacterium transformation of monocotyledonous plants. ADVANTAGE OF THE INVENTION According to the method of this invention, it is possible to produce a transgenic plant more efficiently and much more quickly than the conventional Agrobacterium transformation method.

[0013]

SUMMARY OF THE INVENTION The present invention relates to a method for transforming monocotyledonous plants, comprising the step of infecting intact seeds with Agrobacterium containing the desired recombinant gene. In the method of the present invention, the seed is infected in an intact state, and no treatment such as dedifferentiation of the plant sample to be transformed is required.

The seed subjected to infection with Agrobacterium may be a seed 4-5 days after sowing. Also, at the time of infection, the seed may be in a germinated state.

The monocotyledon plant to be transformed is preferably a grass plant, more preferably a rice plant (Oryza).
sativa L. ).

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

The "plant" to which the method of the present invention is applied is a monocotyledonous plant. Preferred monocotyledonous plants include grassy plants (eg, rice and corn). The most preferred plant to which the method of the present invention is applied is rice, especially japonica rice. Also "plant"
Means a plant and seeds obtained from the plant, unless otherwise indicated.

(Preparation of Plant Expression Vector) In order to introduce a desired recombinant gene into monocotyledonous plants, an appropriate plant expression vector containing the desired recombinant gene is constructed.
Such a plant expression vector can be produced using a gene recombination technique well known to those skilled in the art. For construction of a plant expression vector for use in the Agrobacterium transformation method, for example, a pBI-based vector is suitably used, but is not limited thereto.

"A desired recombinant gene" refers to any polynucleotide desired to be introduced into a plant.
The desired recombinant gene in the present invention is not limited to one isolated from nature, and may also include a synthetic polynucleotide. The synthetic polynucleotide can be obtained, for example, by synthesizing or modifying a gene whose sequence is known by a method well known to those skilled in the art. Examples of the desired recombinant gene in the present invention include, for example, any polynucleotide that is desired to be expressed in a transformed plant and is endogenous or exogenous to the plant, and an endogenous gene in a plant. When expression control is desired, a polynucleotide containing the antisense sequence of the target gene can be used.

If expression in a plant is intended, the desired recombinant gene will include its own promoter (ie, the promoter to which it is operably linked in nature) in an operable manner, or When no promoter is contained or when it is desired to further contain a promoter other than its own promoter, it is operably linked to any appropriate promoter. Promoters that can be used include constitutive promoters,
And inducible promoters that are selectively expressed in plants and parts of plants.

[0021] In the plant expression vector, various regulatory elements can be further operably linked in the cells of the host plant. Regulatory elements may suitably include selectable marker genes, plant promoters, terminators, and enhancers. It is well known to those skilled in the art that the type of plant expression vector and the type of regulatory element to be used can vary depending on the purpose of transformation.

The “selectable marker gene” can be used to facilitate selection of a transformed plant. A drug resistance gene such as a hygromycin phosphotransferase (HPT) gene for conferring hygromycin resistance and a neomycin phosphotransferase II (NPTII) gene for conferring kanamycin resistance can be preferably used. It is not limited to.

"Plant promoter" refers to a plant-expressed promoter operably linked to a selectable marker gene. Examples of such promoters include cauliflower mosaic virus (CaMV) 35
S promoter, and nopaline synthase promoter, but are not limited thereto.

"Terminator" is located downstream of the region encoding the protein of the gene,
And a sequence involved in the termination of transcription when transcribed into E. coli and the addition of a polyA sequence. Examples of terminators include:
Examples include, but are not limited to, the CaMV 35S terminator and the nopaline synthase gene terminator (Tnos).

An “enhancer” can be used to increase the expression efficiency of a target gene. As the enhancer, an enhancer region containing an upstream sequence in the CaMV35S promoter is preferable. The enhancer is 1
A plurality may be used per one plant expression vector.

(Transformation of Plant) Agrobacterium used for transformation of monocotyledonous plants may be any bacteria belonging to the genus Agrobacterium, and is preferably Agrobacter.
erium tumefaciens. Agrobacterium is a plant expression vector containing the desired recombinant gene (eg, by electroporation).
Transformed. By infecting a seed with the transformed Agrobacterium, a desired recombinant gene can be introduced into a plant. The introduced recombinant gene is integrated into the genome of the plant. In addition, the genome in a plant includes not only a nuclear chromosome but also a genome contained in various organelles (eg, mitochondria, chloroplasts, etc.) in a plant cell.

The seeds of the plant to be transformed are pre-cultured in an intact state after removing the chaff. “Intact” with respect to seed refers to a state in which the seed has not been subjected to artificial manipulations such as removing ovules and damaging the scutellum.

In the pre-culture, the seeds are grown in a medium (eg, 2,4-D) containing an appropriate concentration of auxin (eg, 2,4-D).
N6D medium), typically for 4-5 days,
It can be kept warm, preferably for 5 days. The preculture is completed before the seed tissue enters the dedifferentiation process. The temperature at this time is typically 25 to 35 ° C, preferably 27 to 32 ° C.
It is. After the completion of the preculture, the seeds are sterilized and then thoroughly washed with water. The seed can then be infected with the transformed Agrobacterium under aseptic operation.

Agrobacterium infection (co-culture)
During, the seeds are in the dark, typically for 2-5 days,
It is preferably kept warm for 3 days. The temperature at this time is typically 26 to 28 ° C, preferably 28 ° C. The seed is then subjected to treatment with a suitable disinfectant (eg, carbenicillin) to disinfect Agrobacterium in the medium. Transformed seeds are selected based on a selection marker (for example, drug resistance such as hygromycin resistance).

After culturing under appropriate sterilization and selection conditions, the selected transformed seeds are transferred to a regeneration medium (for example, MS medium) containing an appropriate plant regulator, and incubated for an appropriate period. Can be done. In order to regenerate a plant, the redifferentiated transformant is transferred to a rooting medium (for example, an MS medium without a plant regulator). After root development has been confirmed, the transformants can be potted.

The desired recombinant gene introduced into the plant is
It can act for the intended purpose in the plant (eg, expression of a new trait of interest, or regulation of expression of some endogenous gene).

Whether or not a desired recombinant gene has been introduced into a plant can be confirmed using techniques well known to those skilled in the art.
This confirmation can be performed, for example, using Northern blot analysis. Specifically, total RNA is extracted from the leaves of the regenerated plants, electrophoresed on denatured agarose, and then blotted to an appropriate membrane. By hybridizing the blot with a labeled RNA probe complementary to a part of the transgene, mRNA of the gene of interest can be detected. Alternatively, if regulation of the expression of the endogenous gene in the plant is desired by introduction of the desired recombinant gene, expression of the target endogenous gene can be tested, for example, using Northern blot analysis as described above. If the expression of the target endogenous gene is significantly suppressed relative to its expression in a non-transformed control plant, then the desired recombinant gene has been introduced into the plant and has affected expression. It is confirmed.

The conventional method requires a dedifferentiation induction period of usually 3 to 4 weeks before infection with Agrobacterium. In contrast, the method of the present invention does not require a step of inducing dedifferentiation, so that it is possible to reduce the number of days required to produce a transformed monocotyledonous plant. Further, according to the method of the present invention, the time required for selection in the conventional method can be shortened, and the influence of culture mutation can be reduced.

In one preferred embodiment of the method of the present invention, the number of days required to produce a transformed monocotyledonous plant is about 50 days, and a conventional Agrobacterium transformation method (for example, Less than about two-thirds of the number of days required (see Example 2) (about 90 days). Further, according to the method of the present invention, a transformation efficiency of 10 to 15% can be obtained in the case of Nipponbare seeds. Similar high transformation efficiencies can be achieved with other rice varieties such as Dotonkoi and Kitaake. Therefore, by using the method of the present invention, it is possible to produce a transformed plant more efficiently and more quickly than conventional transformation methods.

[0035]

EXAMPLES The present invention will be specifically described below with reference to examples. This example does not limit the invention. Materials, reagents, etc., used in the examples are available from commercial sources unless otherwise specified.

(Example 1: Transformation of rice plant by the method of the present invention) Nipponbare seeds, a representative rice variety, were
After removal of the rice husks, they were sterilized in a 2.5% sodium hypochlorite (NaClO) solution in an intact state. After sufficient washing with water, the rice was subjected to the following aseptic operation.

(Preculture) Seeds were cultured in N6 containing 2,4-D.
D medium (30 g / l sucrose, 0.3 g / l casamino acid, 2.8 g / l proline, 2 mg / l 2,4-D,
4 g / l gel light, pH 5.8) and inoculated for 5 days.
The temperature was kept at 27 ° C to 32 ° C. During this time the seeds germinated (FIG. 1).

(Plant Expression Vector) As a plant expression vector for transforming Agrobacterium, a plasmid in which the GUS gene containing the first intron of the castor catalase gene and the hygromycin resistance gene are linked. Using pIG121Hm (Nakamura et al., Plant Biotechnology II, Hyundai Chemical Special Edition, p.
p. 123-132 (1991)). pIG121Hm
Was used to transform Agrobacterium EHA101 (Hood et al., J. Bacteriol., 16).
8: 1291-1301 (1986)). EHA101
Is a bacterium in which the vir region of the helper plasmid is derived from the strongly pathogenic Agrobacterium A281.

(Agrobacterium infection) The above-mentioned seeds pre-cultured were immersed in a suspension of the transformed Agrobacterium, and then immersed in 2N6-AS medium (30 g / l sucrose, 10 g / l glucose, 0.3 g / L casamino acid,
2 mg / l 2,4-D, 10 mg / l acetosyringone, 4 g / l gellite, pH 5.2).
The cells were co-cultured in the dark at 28 ° C. for 3 days.

(Eradication and Selection) After the completion of the coculture, 5
Agrobacterium was washed from the seeds using N6D medium containing 00 mg / l carbenicillin. Next, the transformed seeds were selected under the following conditions. First round : 2 supplemented with carbenicillin (500 mg / l) and hygromycin (25 mg / l)
Seeds were placed on N6D medium containing mg / l 2,4-D and kept at 27 ° C. to 32 ° C. for 7 days. Second round of selection : supplemented with carbenicillin (500 mg / l) and hygromycin (25 mg / l), 2
Seeds were placed on N6D medium containing 4 mg / l 2,4-D and kept at 27 ° C. to 32 ° C. for an additional 7 days.

(Regeneration) The transformed seeds thus selected were regenerated under the following conditions. First regeneration : regeneration medium (carbenicillin (50
0 mg / l) and hygromycin (25 mg / l)
Medium (30 g / l sucrose, 30 g /
(1 sorbitol, 2 g / l casamino acid, 2 mg / l kinetin, 0.002 mg / l NAA, 4 g / l gellite, pH 5.8), and the selected seeds were placed and kept at 27 ° C to 32 ° C for 2 weeks. . Second regeneration : Using the same regeneration medium used in the first regeneration, for an additional 2 weeks at 27 ° C.
Incubated at ~ 32 ° C.

(Picked up) The redifferentiated transformant was added to a rooting medium (supplemented with hygromycin (25 mg / l).
(Hormone-free MS medium), and after confirming the growth of the roots, they were lifted (FIG. 2).

Example 2 Transformation of Rice Plant by Conventional Method For comparison with the method described in Example 1, rice plants were transformed by a conventional method using Nipponbare as a material for transformation. Transformation was performed as follows.

(Callus Induction) Nipponbare seeds were sterilized after removal of the rice husks, and were seeded on a callus induction medium (N6D medium containing 2 mg / l of 2,4-D). Incubated at ° C. Approximately 4 weeks after the start of callus induction, the proliferated callus derived from the scutellum was used for transformation.

(Transformation) The obtained calli were infected with Agrobacterium EHA101 transformed with the plant expression vector pIG121Hm as described in Example 1 and transformed on a 2N6-AS medium in the dark for 3 hours. 28 ° C for days
And co-cultured.

(Eradication and Selection) Using N6D medium containing 500 mg / l carbenicillin,
Agrobacterium was washed away. Next, the transformed calli were selected under the following conditions. First round of selection : 2 m supplemented with carbenicillin (500 mg / l) and hygromycin (50 mg / l)
The calli were placed on N6D medium containing g / l 2,4-D and kept at 27 ° C to 32 ° C for 2 weeks. Second selection : 2- supplemented with carbenicillin (500 mg / l) and hygromycin (50 mg / l)
The calli were placed on N6D medium containing 4 mg / l 2,4-D and kept at 27 ° C. to 32 ° C. for another two weeks.

(Regeneration, Rooting and Potting) The selected transformed seeds were regenerated under the same conditions as in Example 1,
We went up to the pot.

(Results) FIG. 3 shows a comparison between an example of transformation by the conventional method and an example of transformation by the method of the present invention. After seeding, the number of days required for the transformant to be raised was about 90 days in the conventional method, but about 50 days in the method of the present invention (FIG. 3).
(A)). Comparing about 50 days after seeding, the transformant in the method of the present invention was in a state capable of being potted, whereas the transformant in the conventional method was still in the process of regeneration ( FIG. 3 (b). In summary, the implementation of the method of the present invention reduced the time required for transformation to about two thirds or less of the conventional method.

[0049]

According to the present invention, there is provided an improved method for Agrobacterium-mediated monocot transformation. In the method of the present invention, intact seeds of a plant intended to be transformed are infected with Agrobacterium containing the desired recombinant gene. The use of the present invention makes it possible to produce a transformed plant more efficiently and more quickly.

[Brief description of the drawings]

FIG. 1 is a photograph showing the morphology of an organism, showing the state of rice seeds immediately before infection with Agrobacterium.

FIG. 2 is a photograph showing the morphology of an organism showing a redifferentiated individual of rice obtained by the method of the present invention about 50 days after sowing.

FIG. 3 (a) is a photograph showing the morphology of an organism, comparing a transformant according to the conventional method about 90 days after seeding with a transformant according to the method of the present invention about 50 days after seeding. .
(B) a transformant according to a conventional method about 50 days after seeding,
1 is a photograph showing the morphology of an organism as compared with a transformant according to the method of the present invention about 50 days after seeding.

[Procedure amendment]

[Submission date] February 25, 2000 (200.2.2
5)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Masashi Ugaki 2-1-2 Kannondai, Tsukuba, Ibaraki Pref. Agriculture, Forestry and Fisheries National Institute for Agricultural and Biological Resources (72) Inventor Fumio Shiohara 2-1 Kannondai, Tsukuba, Ibaraki -2 Agriculture, Forestry and Fisheries Research Institute of Agricultural and Biological Resources (72) Inventor Naoto Shibuya 2-1-2 Kannondai, Tsukuba-shi, Ibaraki Prefecture 2-1-2 Inside the Agricultural and Biological Resources Institute, Ministry of Agriculture, Forestry and Fisheries (72) Inventor Kazuko Ono 2-1-2, Kannondai, Tsukuba City, Ibaraki Prefecture Inside the Agricultural Biological Resources Institute, Ministry of Agriculture, Forestry and Fisheries (72) Inventor Akemi Tagiri Ibaraki 2-1-2 Kannondai, Tsukuba, Japan Prefectural Agriculture, Forestry and Fisheries National Institute for Agricultural and Biological Resources (72) Inventor Yaeko Nishizawa 2-1-2 Kannondai, Tsukuba, Ibaraki Hayashimizu ministry NIAS in the F-term (reference) 2B030 AA02 AB03 CA06 CA17 CA19 CB02 CD03 CD06 CD09 CD13 CD17 4B024 AA08 BA80 CA03 DA01 DA05 EA04 EA10 GA11 HA01 4B065 AA11X AA11Y AA89X AA89Y AB01 BA02

Claims (5)

[Claims] 1. A method of transforming a monocotyledonous plant, comprising infecting an intact seed with an Agrobacterium containing a desired recombinant gene. 2. The method according to claim 1, wherein the seed is a seed 4 to 5 days after sowing. 3. The seed according to claim 1, wherein said seed is a germinated seed.
Or the method of 2. 4. The monocotyledonous plant is a gramineous plant,
A method according to any of claims 1 to 3. 5. The method according to claim 4, wherein the gramineous plant is rice.