JP2000093028A - Enhancement of fungal disease resistance of grape - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enhance the resistance of grapes to powdery mildew and anthracnose by transferring a chitinase gene into the adventitious embryos of grapes followed by selecting the resultant transformed adventitious embryos which are then regenerated in the plant. SOLUTION: The resistance of grapes to fungal diseases including powdery mildew and anthracnose is enhanced by transferring a chitinase gene derived from pref. rice into the adventitious embryos acquired from the tissue of grapes pref. as a variety of cultivated grapes followed by selecting the resultant transformed adventitious embryos which are then regenerated in the plant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for enhancing the resistance of grape to fungal diseases.

[0002]

2. Description of the Prior Art Grapes have been known to be affected by many fungal infections. As a method for imparting resistance to fungal diseases to grapes, there is known a method in which the grape is crossed with resistant grape and a resistant individual is selected from the hybrid seedlings. However, in the method of crossing with resistant grape and selecting resistant individuals from the hybrid seedlings, properties other than disease resistance are also introduced,
It was not possible to confer only disease resistant properties. Further, there are often no grapes that are resistant to a particular disease. Further, when the resistance property is a recessive trait or when a plurality of loci are involved, at least two or more crossings and selfing are required.

[0003]

An object of the present invention is to find a method for enhancing the resistance of grape to fungal diseases.

[0004]

Means for Solving the Problems In view of the above-mentioned situation, the present inventors have conducted intensive studies to find a method for enhancing fungal disease resistance of grapes, and as a result, have obtained an uncertainty obtained from grape tissues. The present inventors have found a method for enhancing fungal disease resistance of grapes, which comprises introducing a chitinase gene into an embryo, selecting a transformed somatic embryo, and then regenerating the plant body, thereby completing the present invention.

That is, the present invention provides (1) a method of introducing a chitinase gene into a somatic embryo obtained from a grape tissue, selecting a transformed somatic embryo, and regenerating the plant into a grape. A method for enhancing fungal disease resistance,
(2) the method according to (1), wherein the chitinase gene is a rice-derived chitinase gene; (3) the method according to (1), wherein the grapes are cultivated grape varieties; (4) the ovules whose grape tissue is unfertilized (5) The method according to (1), wherein the grape tissue is a leaf.

In the present invention, first, a somatic embryo is induced from a grape tissue. The somatic embryo here refers to an embryo having a heart-shaped, torpedo-shaped, or spherical-shaped appearance, which can be regenerated into a plant body, and in which buds and root primordia are often observed. As a condition for inducing an adventitious embryo, for example, a medium for culturing a plant tissue with a carbon source such as sucrose or a plant hormone may be used. In addition, although somatic embryos can be directly induced from grape tissues, in general, somatic embryos are often induced once, and then induced. As a medium for plant tissue culture, for example, Murashige and Skoog's medium (MS medium, Mu
rashige et al. 1962. Physiol. Plant. 15: 473-49
7), Rinsmeyer and Skoog's medium (LS medium),
Examples include a Gamborg's medium (B5 medium) and a niche-to-niche medium (NN medium). It is also effective to change the contents of the constituent components, add or delete vitamins and amino acids as necessary. .

Examples of plant hormones include indoleacetic acid, indolebutyric acid, 2,4-dichlorophenoxyacetic acid, 4-chlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, 4-fluorophenoxyacetic acid, naphthaleneacetic acid, Auxins such as naphthoxyacetic acid and picloram, zeatin, 6-benzylaminopurine, kinetin, isopentenyladenine, 2-chloro-4-
Cytokinins such as pyridyl-phenylurea (4PU) and 1,2,3-thiadiazol-5-yl-phenylurea (TDZ) can be mentioned.

[0008] The grape tissue for inducing somatic embryos may be anything as long as it can induce somatic embryos, such as unfertilized ovules, anthers, pollen, fertilized embryos and ovary, leaves, stems, roots and the like. Tissues may be mentioned, but unfertilized ovules and leaves are particularly effective. The grape in the present invention is a genus Vitis.
Genus), for example, Vitis vi
nefera L., Vitis labrusca L., Vitis amurensis Rup
r., Vitis berlandieri Phanchon, Vitis californica
Bentham, Vitis girdiana Munson, Vitis rupestris Sc
heele, Vitis rotundiforia Michaux, Vitis thunbergi
i Sieb. et Zucc., Vitis shiragai Makino, Vitis coi
gnetiae Pulliat, Vitis flexuosa Thunb., Vitis kius
iana Momiyama and the like.

Further, the genus Vitis includes cultivated grapes (Vitis v
inefera L., Vitis labrusca L.), and cultivated grape varieties, for example, Kyoho, Muscat berry A, Neo Muscat, Pione, Kaiji, Takao, Hiro Hamburg, Olympia, Ryuho, Benizuho, Minoru Fuji, King De La, Rosario Bianco, Aki Queen, No Red, Honey Sidless, Koshu Sanshaku, Cabernet Sauvignon, Campbell Early, Mario, Pinonoar, Chadonnay, Merlot Blanc, Emperor, Vilad Blanc, Merlot and the like. Next, the chitinase gene is introduced into the somatic embryo obtained from the grape tissue.
Agrobacterium tumefaci
ens and Agrobacterium rhizogenes, and a method using a gene gun.
Methods using tumefaciens or Agrobacterium rhizogenes are more efficient.

For example, in the method using Agrobacterium tumefaciens, first, a plasmid of a binary vector containing a chitinase gene is introduced into Agrobacterium tumefaciens by a tripartal mating method, an electroporation method, or a direct introduction method. Then, the somatic embryo obtained from the grape tissue is coexistent and infected with Agrobacterium tumefaciens containing a chitinase gene, and cultured for several hours to several days in a medium containing no antibiotics. Then, the medium for culturing the somatic embryos is supplemented with an antibiotic for eliminating Agrobacterium tumefaciens such as claforan and carbenicillin, and a compound for selecting transformed somatic embryos such as kanamycin or geneticin or hygromycin. The culture is continued for at least one month, and the somatic embryo into which the desired chitinase gene has been introduced is selected.

In the process of selecting transformed somatic embryos, Agrobact
There is a case where a new transformed somatic embryo is generated from the surface of the somatic embryo infected with erium, and a case where the first somatic embryo is selected as it is. As long as the desired chitinase gene has been introduced, either method may be used. Next, the transformed somatic embryo is regenerated into a plant on a regeneration medium to obtain a young plant. Then, it is acclimated and potted by a usual method to obtain a grape plant into which a chitinase gene has been introduced.
The chitinase gene used can be of any origin, but is more preferably of plant origin. Furthermore, a chitinase gene derived from rice (Nishizawa et al. 1
993. Mol. Gen. Genet. 241: 1-10) is effective. Grape plants into which the chitinase gene has been introduced can enhance resistance to fungal diseases.

Examples of fungal diseases of grapes include black mold, black mold, rust, gray mold, downy mildew and the like. In addition, when the chitinase gene was introduced, by linking to various promoters, the expression of a strong chitinase gene, the expression of a site-specific chitinase gene, the expression of a chitinase gene by stimulation, etc., were further translated from the introduced chitinase gene. It is possible to manipulate where the chitinase protein is located.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0014]

Example 1 (Induction of Adventitious Embryo from Leaf of Koshu Sansho Grape Varieties) A leaf disk of a fully developed leaf of a grape variety "Koshu Sansha" cultivated in a greenhouse was used. Sodium hypochlorite aqueous solution with a few drops of Tween20 (effective chlorine content about 0.5%
Was diluted for about 15 minutes, and then washed three times with sterile distilled water. Middle rib removed, diameter 5
Leaf discs were prepared with a sterile cork borer of 0.2 mm and inoculated on callus induction medium with the back of the leaf up.
The fresh weight of the leaf disc punched with a cork borer was about 3.8 mg on average. As a callus forming medium, 2% sucrose, 0.8% agar, 5 μM 2,4
-Dichlorophenoxyacetic acid (2,4-D), 10 μM 6-benzylaminopurine (BA) was added, and the pH was 5.8.
A niche and a niche medium (NN medium) adjusted to 0 were used. The cells were cultured in the dark at 28 ° C. for several months to form callus and adventitious embryos.

[0015]

Example 2 (Induction of somatic embryos from unfertilized ovules of grape variety Neo Muscat) Grape variety "Neo Muscat"
Unfertilized ovules were aseptically removed from flower buds 3 to 10 days before flowering. As a callus formation medium, 3% sucrose, 1μ
M 2,4-D and 0.2 μM 2-chloro-4-pyridyl-phenylurea (4PU) were added, and an NN liquid medium adjusted to pH 5.8 was used. 80 rotations of unfertilized ovules /
The cells were cultured with shaking at 25 ° C. in the dark for 25 minutes to form calli. The formed calli were subcultured in a liquid medium of the same composition. The subcultured calli were transplanted to a Gamborg's medium (B5 medium) or MS medium containing no hormone to obtain adventitious embryos.

[0016]

Example 3 (Introduction of Chitinase Gene) Agrobacterium tumefaciens L
Plasmid pBI121-RCC2 (see FIG. 1) containing a rice-derived chitinase gene was introduced into strain BA4404. pBI121-RCC2 is composed of a rice chitinase gene (RCC2) linked between a cauliflower mosaic virus-derived 35S promoter (P35S) and a nopaline synthetase-derived terminator (TNOS), a nopaline synthetase-derived promoter (PNOS), and a terminator (TNOS). ) Has a kanamycin resistance gene (NPTII, neomycin phosphotransferase).

Using somatic embryos of grape varieties Koshu Sansho and Neo Muscat, the gene was introduced into the somatic embryos by the agro method. LBA4404 having pBI121-RCC2 was added to an LB medium (25 mg / l rifampicillin, 250 mg / l streptomycin, 50 mg) for 24 hours at 28 ° C.
/ L of kanamycin). After culture, 60
The cells were collected by centrifugation at 00 rpm for 5 minutes, and the supernatant was removed.
The cells were suspended in an MS hormone-free medium at pH 5.2 containing 0 μM acetosyringone. Grape somatic embryos with LBA440
4 for about 15 minutes, then remove excess liquid with sterile filter paper and remove hormone-free MS medium (3% sucrose,
It was transplanted to pH 5.2) containing 0.8% agar. After 3 days, the somatic embryos were washed with 50 mg / l kanamycin and 200 m
The cells were transferred to a selection medium (MS, 3% sucrose, 0.8% agar, pH 5.80) containing g / l claforan. Approximately every one month, somatic embryos were transferred to a selection medium of the same composition. After about 3 months, transformed somatic embryos resistant to kanamycin were obtained.
The adventitious embryos were redifferentiated in a selection medium of the same composition, rooted, acclimated and potted.

[0018]

Example 4 (Confirmation of introduced genes) From the fully expanded leaves of 11 neo-Muscat transformed plants and non-transformed plants that had been acclimated and raised, all DNs were obtained by the CTAB method.
A was extracted. The transgene was confirmed by PCR. The primer used was 5'-TGGATCCAGCGGCTCGTCGGTTG
-3 'and 5'-GTATAATTGCGGGACTCTAAT-3', 5'-CCCCTCGGTATCC
With AATTAGAG-3 'and 5'-CGGGGGGTGGGCGAAGAACTCCAG-3, the former primer combination detects the RCC2 gene, and the latter combination detects the NPTII gene.

DNA extracted from about 10 ng of grape, 10 pmole of each primer, 1.5 mM MgC in final concentration
The PCR reaction was carried out under the conditions of a PCR buffer containing l, 4 mM bases of 0.2 mM each, and 5 units of Taq Polymerase (Perkin Elmer). The liquid volume was 20 μl. The reaction temperature condition is as follows when RCC2 gene is detected.
One cycle of 94 ° C for 5 minutes, 55 ° C for 1 minute, 72 ° C for 2 minutes, 9
35 cycles of 4 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 2 minutes, 9
One cycle of 4 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 10 minutes was performed. In the case of NPTII gene detection, 94 ° C for 5 minutes, 62 ° C for 1 minute
1 minute, 72 ° C for 2 minutes, 94 ° C for 1 minute, 62 ° C for 1 minute
35 cycles of 2 minutes at 72 ° C, 1 minute at 94 ° C, 1 minute at 62 ° C
For 1 minute and 72 ° C. for 10 minutes. As a result of analyzing the PCR product by electrophoresis, rice-derived chitinase RCC2 gene and NPTII gene were detected in all the transformed individuals.

[0020]

Example 5 (Determination of disease resistance to black rot) Nine transformants of the obtained Neo-Muscat transformant were used. Black rot (pathogen: Elsinoe ampel) using 9 isolated leaves
Ina) resistance test was performed. A suspension of black rot spores (approximately 104 / ml) was dropped on the collected leaves of the second and third leaf positions, kept under humid conditions for 1 day, and then placed in an isolated glass room at 25 ° C. Thirteen days later, the morbidity index of the inoculation site (no lesions: 0 to 2: 5) was examined. Table 1 shows the results. A lower morbidity index was observed in all transformed individuals than in non-transformed individuals.

[0021]

[Table 1]

[0022]

Example 6 (Disease resistance test against powdery mildew) Eight Neo-Muscat transformants obtained were cultured at 4 ° C for about 4 days.
After low-temperature treatment, cutting clones were prepared. Using cuttings cloned seedlings, powdery mildew (pathogen: Uncinula neca
tor) test was performed. Spraying and inoculating conidia (approximately 104 / ml) on new leaves, keeping them under humid conditions for 1-2 days, placing them in an isolated glass room at 25 ° C, and after 19 and 30 days, the number of lesions per leaf and The morbidity index (no lesions: 0 to complete: 3) was investigated. Table 2 shows the results. Transformants NM-Chi-1, NM-Chi-11, NM-
Chi-17, NM-Chi-19 and NM-Chi-20 were observed to have fewer lesions per leaf 19 days after inoculation than non-transformants.

[0023]

[Table 2]

[0024]

Example 7 (Chitinase RCC with Promoter Replacement)
2 Introduction of genes) By tripalental mating method,
Plasmid pBI containing rice-derived chitinase RCC2 gene was added to Agrobacterium tumefaciens strain LBA4404.
121-EN4-RCC2 (see FIG. 1) was introduced. p
BI121-EN4-RCC2 is an enhancer sequence (reference Mitsuhara et al. 1996. Plan) instead of the 35S promoter derived from cauliflower mosaic virus.
t Cell Physiol. 37: 49-59) added to the 35S promoter.

Using somatic embryos of the grape varieties Koshu Sansho and Neo Muscat, genes were introduced into the somatic embryos by the agro method. LBA4 with pBI121-EN4-RCC2
404 in LB medium (25 mg / l rifampicillin, 250 mg / l streptomycin,
(Containing 50 mg / l kanamycin). After the culture, the cells were collected by centrifugation at 6000 rpm for 5 minutes, the supernatant was removed, and M of pH 5.2 containing 100 μM acetosyringone was added.
The cells were suspended in an S hormone-free medium. Grape somatic embryo
Immerse in the A4404 suspension for about 15 minutes, then remove excess liquid with sterile filter paper and remove hormone-free MS medium (3%
It was transplanted to sucrose containing 0.8% agar, pH 5.2). After 3 days, the somatic embryos were transformed with a selection medium containing 50 mg / l kanamycin and 200 mg / l claforan (M
S, 3% sucrose, 0.8% agar, pH 5.80). Approximately three months later, kanamycin-resistant somatic embryos were selected.

[0026]

Example 8 (Introduction of rice-derived chitinase clone RCG3) An Agrobacter
plasmid pBI121-R containing the rice-derived chitinase RCG3 gene in L. ium tumefaciens strain LBA4404.
CG3 (see FIG. 1) was introduced. pBI121-RCG
3 is a plasmid having a rice-derived chitinase clone RCG3 instead of the RCC2 clone. Using somatic embryos of the grape varieties Koshu Sansho and Neo Muscat, the genes were introduced into the somatic embryos by the agro method. pBI121-R
LBA4404 with CG3 was cultured in LB medium (containing 25 mg / l rifampicillin, 250 mg / l streptomycin, 50 mg / l kanamycin) for 24 hours at 28 ° C. After the culture, the cells were collected by centrifugation at 6000 rpm for 5 minutes, the supernatant was removed, and the cells were suspended in an MS hormone-free medium containing 100 μM acetosyringone at pH 5.2. The grape somatic embryo is immersed in the LBA4404 suspension for about 15 minutes, and then the excess liquid is removed with sterile filter paper, and the hormone-free MS medium (containing 3% sucrose, 0.8% agar,
(pH 5.2). After 3 days, the somatic embryo was
g / l kanamycin and 200 mg / l claforan (MS, 3% sucrose, 0.8% agar, p.
H5.80). Approximately three months later, kanamycin-resistant somatic embryos were selected.

[0027]

Reference Example 1 (Introduction of Chitinase Gene Linked with Rubiscos Small Subunit Gene Promoter) pBI
121-ARS-RCC2 (see FIG. 1) is used. Instead of the 35S promoter from the cauliflower mosaic virus, the rubiscosmol subunit gene promoter from Arabidopsis thaliana (Krebbers et al. 1988.
Plant Mol. Biol. 11: 745-759, Ichikawa et al., Breeding 42 (Alternative 2), 1992). It has a rice chitinase clone RCC2 and a kanamycin resistance gene. PBI121-ARS-RCC2 is introduced into Agrobacterium tumefaciens LBA4404 strain.

Using the somatic embryos of the grape varieties Koshu Sansho and Neo Muscat obtained in Examples 1 and 2, a gene is introduced into the somatic embryo by the agro method. pBI121-ARS-
LBA4404 with RCC2 is LB at 30 ° C for 24 hours
Medium (25 mg / l rifampicillin, 250 mg / l
1 streptomycin, 50 mg / l kanamycin). After the culture, the cells are collected by centrifugation at 6000 rpm for 5 minutes, the supernatant is removed, and the cells are suspended in sterile distilled water. The grape somatic embryo is immersed in the LBA4404 suspension for about 15 minutes, and then the excess liquid is removed with sterile filter paper, and the hormone-free MS medium (containing 3% sucrose, 0.8% agar,
(pH 5.80). One day later, somatic embryos were
Selection medium containing MS / mg kanamycin and 200 mg / l claforan (MS, 3% sucrose, 0.8% agar,
pH 5.80). About every one month, the somatic embryo is transferred to a selection medium of the same composition. After about 2-6 months, transformed somatic embryos resistant to kanamycin are obtained. The adventitious embryos are redifferentiated in a selection medium of the same composition, rooted, acclimatized and potted. Thus, site-specific chitinase gene expression is expected.

[0029]

According to the present invention, fungal disease resistance of grape can be enhanced.

[Brief description of the drawings]

FIG. 1. pBI121-RCC2, pBI121-EN
4-RCC2, pBI121-ARS-RCC2, pB
The figure showing I121-RCG3.

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[Procedure amendment]

[Submission date] July 23, 1999 (1999.7.2)
3)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0005

[Correction method] Change

[Correction contents]

That is, the present invention provides (1) a method of introducing a chitinase gene into a somatic embryo obtained from a grape tissue, selecting a transformed somatic embryo, and regenerating the plant into a grape. method of enhancing the powdery mildew or sphaceloma disease resistance, (2) chitinase gene is a chitinase gene from rice (1) the method according, (3) grapes are grown grape varieties (1) according (4) The method according to (1), wherein the grape tissue is an unfertilized ovule.
(5) The method according to (1), wherein the grape tissue is a leaf.

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C12R 1:91) (72) Inventor Tateki Hayashi 5-15-2 Nagayama, Ryugasaki-shi, Ibaraki Prefecture (72) Inventor Nagao Matsuda Ibaraki 1-16-63 Ninomiya, Tsukuba, Fukushima Prefecture F term (reference) BC31 BD50 CA53

Claims (5)

[Claims] The present invention enhances fungal disease resistance of grape, characterized in that a chitinase gene is introduced into a somatic embryo obtained from a grape tissue, a transformed somatic embryo is selected, and the plant is regenerated. how to. 2. The method according to claim 1, wherein the chitinase gene is a rice-derived chitinase gene. 3. The method according to claim 1, wherein the grapes are cultivated grape varieties. 4. The method according to claim 1, wherein the grape tissue is an unfertilized ovule. 5. The method according to claim 1, wherein the grape tissue is a leaf.