JP2012183017A - Japanese pear transformation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a transformation method of a Japanese pear.SOLUTION: The transformation method of the Japanese pear using the agrobacterium method includes sonicating a Japanese pear-derived plant cell or plant part in the presence of an agrobacterium that maintains T-DNA including a transgene, and cocultivating the plant cell or plant part with the agrobacterium using a co-culture medium containing a calcium ion chelator and excluding a calcium ion.

Description

  The present invention relates to a method for transforming Japanese pear.

  Japanese pear is the fourth largest fruit tree in Japan (Ministry of Agriculture, Forestry and Fisheries 2009 Production Agricultural Income Statistics). However, the cultivation of Japanese pears has problems such as “black spot disease” and “black spot disease” as well as “mitsumitsu”, “bad shelf life” and “fruit rust” that significantly reduce the product value of fruits. ing. The research so far has been proceeding with the analysis of candidate gene groups that are considered to be related to these. However, in order to create innovative new varieties by analyzing the functions of the isolated genes and introducing genes, Japanese pears It is necessary to establish a transformation technique capable of stably expressing the transgene. However, no Japanese pear transformation technology has been developed. The main reason for this is that it is very difficult to infect Japanese pears with Agrobacterium.

  In pears, the production of transformants by the Agrobacterium method using adventitious bud formation from pear leaf pieces has been reported (Mourages, F., et al., Plant Cell Rep., 16: 245-249 (1996 )), Using the same method, we have succeeded in producing transformants in the cultivar Lamania and Bartlett. However, transformation by this method has not been successful in Japanese pears, and even pears may have poor transformation efficiency depending on the variety. Therefore, an Agrobacterium-mediated transformation method using a sprout cultured shoot meristem as an explant has been developed for Japanese pear (Japanese Patent Laid-Open No. 2004-283051). However, this method cannot be applied directly to Japanese pears that are difficult to infect with Agrobacterium.

JP 2004-283051 A

Mourages, F., et al., Plant Cell Rep., 16: 245-249 (1996)

  An object of the present invention is to provide a method for transforming Japanese pears.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have cocultured Agrobacterium cells derived from Japanese pears using calcium-free coexisting medium containing EGTA, and sonication. As a result, it was found that high efficiency and stable transformation of Japanese pear can be realized, and the present invention has been completed.

  That is, the present invention includes the following.

  A method for transforming Japanese pears using the Agrobacterium method, wherein the plant cells or plant body parts derived from Japanese pears are sonicated in the presence of Agrobacterium holding T-DNA containing the transgene, and the plant A method comprising coculturing a cell or a plant part with the Agrobacterium using a coexisting medium containing a calcium ion chelator and excluding calcium ions.

  Here, the co-culture medium is preferably prepared using an MS medium or an MS medium having a diluted inorganic salt concentration as a basic medium.

  In a preferred embodiment of this method, the calcium ion chelator is ethylene glycol bis (β-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid.

  In this method, it is also preferred to use Japanese pear-derived plant cells or plant parts derived from cotyledons.

  By using the present invention, a stable transformant of Japanese pear can be produced.

FIG. 1 is a diagram showing the structure of a nucleic acid construct in a binary vector introduced into Agrobacterium for infection with Japanese pear. “Sgfp” is a modified GFP gene for plant introduction (sGFP gene) introduced into Japanese pears in Examples. “35S-pro” represents a cauliflower mosaic virus (CaMV) 35S promoter, and “Nos-pro” and “Nos-ter” represent a nopaline synthase (NOS) promoter and terminator derived from Agrobacterium tumefaciens, respectively. “RB” is a right border arrangement, and “LB” is a left border arrangement. FIG. 2 is a photograph showing the redifferentiation of Japanese pear adventitious buds in which GFP fluorescence was observed. After selection for transformants following co-culture with Agrobacterium (Agrobacterium infection) after 2.5 months of culturing in a regeneration medium containing kanamycin (FIG. 2A), after 4.5 months of culture (FIG. 2B), It is a photograph which shows the external appearance of the redifferentiated individual derived from a cotyledon after 5.5-month culture | cultivation (FIG. 2C). FIG. 3 shows the results of detecting the nucleic acid construct (FIG. 3A) and the GFP gene (FIG. 3B) incorporated into the genome by PCR analysis for genomic DNA extracted from Japanese pear adventitious buds with GFP fluorescence. It is a photograph. “Control” is a sample derived from non-transformed Japanese pear, “Negative control” is a PCR solution prepared without adding genomic DNA (template), and “Plasmid” is a sample derived from a binary vector containing the sGFP gene. FIG. 4 is a photograph showing a Southern blot analysis result of genomic DNA extracted from a Japanese pear adventitious bud individual in which GFP fluorescence was observed. T is a sample derived from a Japanese pear adventitious bud in which GFP fluorescence is observed, and C is a sample derived from a non-transformed Japanese pear.

  Hereinafter, the present invention will be described in detail.

  The present invention relates to a method for transforming Japanese pear based on the Agrobacterium method. In the present invention, when the Agrobacterium method is applied to Japanese pears, the sonication is performed in the presence of Agrobacterium, and further, calcium ions are removed and a coexisting medium containing a calcium ion chelating agent (preferably EGTA). By applying the combined improvement of co-culturing using (co-culture medium), the infection efficiency of Agrobacterium to Japanese pears is greatly increased, and the transformation of Japanese pears becomes possible.

  The Agrobacterium method is a transformation method that is commonly used for gene transfer into plant cells. The Agrobacterium method incorporates its own gene into the genome of an infected plant as T-DNA. It uses the property that can be. In the Agrobacterium method, RB (right border sequence) and LB (left border sequence) held in a plasmid vector or genome of Agrobacterium in plant cells or plant tissues infected with Agrobacterium. The DNA region sandwiched between is separated and is frequently integrated into the genome of plant cells as T-DNA. RB is a 25-base sequence that functions as a starting point for transmission of T-DNA from Agrobacterium into the plant genome. LB is a T-DNA sequence from Agrobacterium to the plant genome. When transmitted, it is a 25-base sequence that functions as an end point for transmission. When introduced into a plant genome, the left end of T-DNA is LB and the right end is RB. The Agrobacterium method can be widely applied to plants that can be infected by Agrobacterium.

  In the present invention, Japanese pear (Pyrus pyrifolia (Burm.) Nakai.) Is targeted for transformation by the Agrobacterium method. In the present invention, the Japanese pear and other pear species (including, but not limited to, Pyrus calleryana Decne), Pyrus betulaefolia Bunge, Pyrus ussuriensis Maxim., Pear ( Pyrus communis L.) etc.) may also be included. Examples of Japanese pears include, but are not limited to, Ninomiya, Natsushizuku, Kosui, Hakuryu, Nana Anjosho, Nana Miyoshi, Hosui, Twentieth Century, Shintaka, Akizuki, Wangshui, etc. Cultivated varieties are listed.

  In the method of the present invention, Agrobacterium carrying T-DNA containing a target gene (transgene) to be introduced into Japanese pear is used for infection. In the present invention, T-DNA is DNA containing RB and LB at both ends and a so-called expression cassette (that is, a nucleic acid construct in which a gene is placed under the control of a promoter and terminator) between them. T-DNA may comprise one or more expression cassettes. In addition to an expression cassette containing a gene (transgene) to be introduced into Japanese pear, T-DNA is a selectable marker gene including a drug resistance gene (for example, kanamycin resistance gene NptII, hygromycin resistance gene, etc.) and / or It is also preferable to include an expression cassette containing a reporter gene such as GUS gene or GFP gene. The expression cassette in T-DNA can be any constitutive promoter or transient promoter (preferably a plant functional promoter capable of inducing gene expression in plant cells, or in bacteria or fungi such as Escherichia coli). For example, constitutive plant functional promoters include cauliflower mosaic virus (CaMV) 35S promoter (Odell JT, et al., Nature, 313: 810-812 (1985)), promoter of nopaline synthase gene derived from Agrobacterium Ti plasmid (Pnos), promoter of ubiquitin gene derived from maize, promoter of actin gene derived from rice, cassava leaf vein mosaic virus (CsVMV) promoter, Tobacco PR1a promoter, Arabidopsis And the PR-1 promoter. Similarly, the expression cassette in T-DNA may comprise any terminator, preferably a plant functional terminator that is functional in plant cells, for example a nopaline synthase (NOS) gene terminator.

  Agrobacterium carrying T-DNA containing the transgene can be prepared by a conventional method. For example, the transgene can be retained on Agrobacterium T-DNA (intermediate vector method). In this case, T-DNA can be contained in the Ti plasmid of Agrobacterium. An intermediate vector incorporating the transgene is introduced into Agrobacterium holding the Ti plasmid using a helper plasmid, and the transgene is incorporated into the T-DNA in the Ti plasmid by homologous recombination. By selecting Agrobacterium that retains the Ti plasmid into which the transgene is incorporated, Agrobacterium that retains the T-DNA containing the transgene can be produced. Details of such an intermediate vector method, which is a gene introduction method using Agrobacterium, are disclosed in, for example, DeBlock, M. et al., EMBO J., 3: 1681-1689 (1984).

  On the other hand, in the binary vector method, which is another gene transfer method using Agrobacterium, the transgene is inserted into the T-DNA of the binary vector, and it is converted into Agrobacterium lacking the Ti plasmid using a helper plasmid. By introducing, Agrobacterium carrying T-DNA containing the transgene can be produced. Details of the binary vector method are described in, for example, de Framond, A., et al., Bio / Technology, 1: 262-269 (1983), Hoekcma, A., et a1., Nature, 303: 179-180 (1983). Deblaere, R., et al., Methods in Enzymol., 153: 277-292, (1987), Rogers, SG, et al., Methodsin Enzymol., 153: 253-277 (1987), etc. Yes.

  Alternatively, T-DNA containing the transgene may be retained in the Agrobacterium genome (chromosome). This method can be carried out, for example, according to Oltmanns, H. et al. (Plant Physiol., 152: 1158-1166 (2010)).

  By infecting a Japanese pear-derived plant cell or plant body part with Agrobacterium having T-DNA containing the transgene, the transgene on T-DNA can be integrated into the plant genome. Thereby, a transformant of Japanese pear can be produced.

  In the present invention, the plant cell or plant part subjected to Agrobacterium infection may be any plant cell or plant part collected and / or prepared from Japanese pear. Here, the Japanese pear-derived plant cell is a cell isolated from any plant organ or plant tissue of Japanese pear (eg, leaf, stem, root, inflorescence, fruit, seed, axillary bud, embryo, pollen, cotyledon, meristem, etc.) The cultured cells, or protoplasts or callus prepared therefrom may be used. The Japanese pear-derived plant part is a plant part (part of the plant) collected from Japanese pear, for example, any plant such as leaves, stems, roots, inflorescences, fruits, seeds, buds, embryos, pollen, or cotyledons. A plant tissue or plant tissue that is an organ or part thereof (eg, meristem, etc.), or is not isolated from a plant body (still constituting a part of the plant body) Also good. The Japanese pear-derived plant part is preferably, for example, a cotyledon (preferably excluding the hypocotyl part) collected from seeds or a part thereof (eg, a slice). The Japanese pear-derived plant part may be an explant (for example, a shoot or a leaf disk) cut from the Japanese pear plant and placed on a medium.

  Infection of Agrobacterium to Japanese pear plant cells or plant body parts can be basically caused by inoculating plant cells or plant body parts with Agrobacterium and then performing co-culture. These treatments are preferably performed under aseptic conditions. Inoculation of plant cells or plant parts with Agrobacterium is usually carried out by bringing the cultured Agrobacterium solution into contact with plant cells or plant body parts. Specifically, the plant cell or plant part may be immersed in the Agrobacterium solution, or the Agrobacterium solution may be added or applied to the plant cell or plant part. The Agrobacterium solution may be prepared by a conventional method. For example, the bacterial solution can be prepared by inoculating Agrobacterium in a YEP medium or YEB medium and culturing at 28 ° C. overnight to 24 hours. Agrobacterium thus cultured is pelleted by centrifuging the culture medium from which calcium ions have been removed (for example, 1/10 MS-Ca + 6.3% sucrose + 3.6% glucose + 10 mM MES (2-morpholino ethane sulfone). Acid) + 0.01% Pluronic F-68 + 100 μM acetosyringone) is preferably used as the Agrobacterium solution.

  In the method of the present invention, plant cells or plant parts are inoculated with Agrobacterium, and then the Japanese pear-derived plant cells or plant parts are sonicated in the presence of the Agrobacterium. The ultrasonic treatment is not limited, and can be performed using, for example, an oscillation frequency of 20 to 50 KHz, preferably 30 to 40 KHz, and more preferably 38 KHz. The ultrasonic treatment is usually performed for 10 seconds or longer, preferably 10 seconds to 30 seconds, more preferably 15 seconds to 25 seconds, for example 20 seconds. The ultrasonic treatment can be performed using, for example, a commercially available ultrasonic cleaner.

  In the method of the present invention, a Japanese pear-derived plant cell or plant part after inoculation with Agrobacterium is used, preferably after the ultrasonic treatment, using a co-culture medium modified by adding a calcium ion chelating agent and removing calcium ions. And co-cultivate with the Agrobacterium. The co-culture medium means a co-culture medium (co-culture medium) that can be used for co-cultivation by the Agrobacterium method. The co-culture medium can be prepared using a plant tissue culture medium as a basic medium. The plant tissue culture medium serving as a basic medium for preparing the co-culture medium is inorganic salts (including vitamins, amino acids and the like). As the basic medium, MS medium (Murashige & Skoog medium), N6 medium, L6 medium, SB medium, R2 medium, Tuleeke medium, SH medium, Knop medium, Tukey & Cox medium, Gamborg B5 medium, Nitsch medium, White medium, LP medium, KNUDSON C medium, and the like, and media in which the inorganic salt concentration is diluted (for example, diluted to 1/2 to 1/20) (medium having a diluted inorganic salt concentration) are included in these media. Is not to be done. As a basic medium for preparing the co-culture medium, an MS medium or an MS medium having a diluted inorganic salt concentration thereof (for example, a 1/2 MS medium in which an MS inorganic salt is diluted to a half concentration, an MS inorganic salt in 1 / 1/4 MS medium diluted to 4 concentrations, 1/10 MS medium diluted with MS inorganic salt concentration to 1/10 concentration, and the like are more preferable. The co-culture medium is composed of a basic medium such as carbon source (sucrose, glucose, etc.), plant hormones such as cytokinins and auxins that function as plant growth promoters (for example, benzyladenine (BA), thidiazuron (TDZ). ), Phenols such as naphthalene acetic acid (NAA), indole acetic acid (IBA)), acetosyringone, surfactants, and agrobacterium methods such as agar for solidification, etc. Can be prepared by appropriately selecting and adding. “Preparing as a basic medium” means preparing a medium (coexisting medium) by adding these additional medium components to the composition of the medium used as the basic medium as necessary.

In the present invention, calcium ion chelating agents such as ethylene glycol bis (β-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid (EGTA), ethylenediamine-N, N , N ', N'-Tetraacetic acid disodium salt dihydrate (EDTA), 1,2-bis (o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid (BAPTA), 1 , 2-bis (o-amino-5′-methylphenoxy) ethane-N, N, N ′, N′-tetraacetic acid tetraacetoxymethyl ester (MAPTAM) and the like are contained. The calcium ion chelating agent (for example, EGTA) is not limited, but is preferably contained in the co-culture medium at a concentration of 0.1 to 100 mM, for example, 1 to 50 mM. Instead of or in addition to calcium ion chelating agents, calcium channel inhibitors (verapamil, diltiazem), H ⁺ -ATPase inhibitors (vanadic acid), calmodulin inhibitors (W- 7) Protein kinase inhibitors (staurosporine) can also be used.

  In the present invention, calcium ions are further excluded from the composition of the co-culture medium used for co-culture (the co-culture medium does not contain calcium ions). Since the basic medium used for the preparation of the coexisting medium contains inorganic salts, the coexisting medium excluding calcium ions can be prepared by eliminating calcium ions in the inorganic salts. The removal of calcium ions can be performed, for example, by preparing a medium without adding a salt containing calcium ions such as calcium chloride in inorganic salts.

  In the method of the present invention, cocultivation of Agrobacterium with Japanese pear-derived plant cells or plant parts subjected to ultrasonic treatment is performed using the above-mentioned predetermined co-culture medium. The conditions for co-cultivation can be appropriately adjusted by those skilled in the art. For example, co-cultivation can be performed at a temperature of 18 to 28 ° C, preferably 24 to 27 ° C, more preferably 26 ° C. More preferably, the co-culture is performed in the dark. The coculture is not limited, but may be performed, for example, for 1 to 10 days, preferably 3 to 7 days, more preferably 4 to 6 days.

  Conventionally, it has been suggested that in hypericum perforatum, which is difficult to transform, the plant side may have a protective reaction based on a kind of oxidation reaction called “oxidative burst” against Agrobacterium infection (Franklin, G. et al., Planta, 227: 1401-1408 (2008)). In the present invention, calcium ion chelating agents (EGTA, etc.) contained in the co-culture medium can suppress the inflow of calcium ions into cells and suppress the protective reaction such as oxidative burst against Agrobacterium infection of Japanese pear. it can. Furthermore, by removing calcium ions from the co-culture medium, the intracellular influx of calcium ions is suppressed, and defense reactions such as oxidative burst are suppressed. As a result, the infection efficiency of Agrobacterium is remarkably increased and transformation is induced.

  After the co-culture is completed, it is preferable that the Japanese pear-derived plant cell or plant part is washed with a culture medium, then re-differentiated by subculture using a regeneration medium, and grown into a plant body. The culture in the regeneration medium is not limited, but it is preferably performed under dark conditions, for example, 18 ° C to 28 ° C. Regeneration media include plant culture media such as MS media, plant hormones (but not limited to NAA, BA, TDZ, etc.), antibiotics for killing Agrobacterium (carbecillin, claphoran, etc.) and transformation. A medium supplemented with antibiotics (kanamycin, hygromycin, etc.) for selecting a body can be used. For the redifferentiated individuals thus obtained, the transformation rate can also be determined by confirming the trait (for example, fluorescence) produced by transformation and calculating the ratio at which the trait was observed. The redifferentiated individuals in the present invention include redifferentiated plants, adventitious bud tissues, adventitious roots, tissues derived from these transplanted tissues, and the like. According to the transformation method of the present invention, for example, the average transformation rate after 2 weeks from the start of co-cultivation is 40% or more, more preferably 60% or more, still more preferably 75% or more, and particularly preferably 85%. This can be done. In the transformation method of the present invention, for example, the transformation rate after 2 weeks from the start of co-culture is preferably 1.5 times or more, compared to the case of non-sonication using EGTA-free and calcium-containing medium. Preferably, it is increased to 3 times or more, more preferably 5 times or more, still more preferably 10 times or more, and particularly preferably 25 times or more. Further, in the transformation method of the present invention, the transformant can be maintained at a higher rate over a longer period (for example, 5 months or more).

  According to the method of the present invention, it becomes possible to produce a Japanese pear transformant based on sonication and suppression of intracellular entry of calcium ions. The present invention also provides Japanese pear transformants (including Japanese pear transformed plants, transformed cells, and transformed plant parts and tissues) obtained by the transformation method according to the present invention.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

1. Preparation of Agrobacterium Binary vector pBIN19-sgfp (Chiu, WL et al., Current Biol) containing nucleic acid construct (expression cassette; Fig. 1) containing kanamycin resistance gene (NPTII) and GFP gene (green fluorescent protein coding gene) 6: 325-330 (1996), Heim, R. et al., Nature. 373: 663-664 (1995)) and Agrobacterium LBA4404 strain (Ooms, G. et al., Plasmid, 7: 15- 29 (1982)) to produce Agrobacterium GFP / LBA4404 used for Japanese pear transformation. In addition, the vector pIG121 (Ohta, S. et al., Plant Cell Physiol. 31) containing the kanamycin resistance gene (NPTII) and hygromycin resistance gene and the β-glucuronidase gene (GUS) in which the castor catalase gene intron is connected to the 35S promoter. : 805-813 (1990)) was introduced into Agrobacterium strain LBA4404 to produce Agrobacterium pIG121 / LBA4404.

Agrobacterium pIG121 / LBA4404 or GFP / LBA4404 was cultured overnight at 28 ° C. and 140 rpm in 3 ml YEB medium containing 50 μg / ml kanamycin, 50 μg / ml rifampicin, 300 μg / ml streptomycin. The cultured Agrobacterium solution was centrifuged at 6000 rpm at room temperature, and the supernatant was removed. The obtained Agrobacterium pellet was added to the medium (1/10 MS-Ca + 6.3% sucrose + 3.6% glucose + 10 mM MES (2-morpholinoethanesulfonic acid) + 0.01% pluronic F-68 + 100 μM acetosyringone ( Resuspended to pH 5.4 or pH 5.8)), the concentration of Agrobacterium was adjusted to 10 ⁸ cfu / ml. This Agrobacterium suspension was used for infection.

2. Effect of EGTA on the efficiency of Agrobacterium infection in cultured stem sections For cultivars of Japanese pear (Pyrus pyrifolia (Burm.) Nakai.), Agar medium [MS medium (Murashige, T. and Skoog, F., Physiol. Plant., 15: 473-497 (1962); potassium nitrate 1900mg / L, ammonium nitrate 1650mg / L, potassium dihydrogen phosphate 170mg / L, magnesium sulfate heptahydrate 370mg / L, calcium chloride dihydrate 440mg / L, boric acid 6.2mg / L, manganese sulfate monohydrate 16.9mg / L, zinc sulfate heptahydrate 8.6mg / L, sodium molybdate dihydrate 0.25mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, potassium iodide 0.83 mg / L, myo-inositol 100 mg / L, glycine 2 mg / L, nicotinic acid 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, hydrochloric acid Thiamine 0.1mg / L, Ethylenediamine-N, N, N ', N'-Tetraacetic acid disodium salt dihydrate 37.3mg / L, Ferrous sulfate heptahydrate 27.8mg / L, Claus was 30g / L) + 0.1μM NAA (naphthalene acetic acid) + 10 [mu] M BA (benzyladenine) + 1 [mu] M GA ₃ (to form a chute by culture in gibberellic acid) + 0.85% agar (pH 5.8)]. The leaves were cut off from the shoots, and the stem portions were cut out with a length of 3 to 5 mm. The cut stem sections were immersed in a cutting medium (1/10 MS-Ca + 6.3% sucrose + 3.6% glucose + 10 mM MES (2-morpholinoethanesulfonic acid) (pH 5.8)). As a control, cultured stem sections were similarly prepared from pear (Pyrus communis L.) cultivars La France and Bartlett.

  The cultured stalk section thus prepared was immersed in the Agrobacterium suspension (pIG121 / LBA4404) prepared above. After this Agrobacterium infection treatment, the culture stem section was taken out from the Agrobacterium suspension and added with 3% sucrose + 10 mM MES + 100 μM acetosyringone + 5 μM NAA + 10 μM BA + 0.85% agar, 1/10 MS medium, 1/10 The cells were transplanted to MS-Ca medium, 1/10 MS + EGTA medium, or 1/10 MS-Ca + EGTA medium, and co-cultured in the dark at 26 ° C. for 5 days.

  The composition of the 1/10 MS medium used in the examples is as follows: potassium nitrate 190 mg / L, ammonium nitrate 165 mg / L, potassium dihydrogen phosphate 17 mg / L, magnesium sulfate heptahydrate 37 mg / L, calcium chloride Hydrate 44mg / L, Boric acid 0.62mg / L, Manganese sulfate monohydrate 1.69mg / L, Zinc sulfate heptahydrate 0.86mg / L, Sodium molybdate dihydrate 0.025mg / L, Copper sulfate Pentahydrate 0.0025mg / L, cobalt chloride hexahydrate 0.0025mg / L, potassium iodide 0.083mg / L, myo-inositol 10mg / L, glycine 0.2mg / L, nicotinic acid 0.05mg / L, pyridoxine hydrochloride 0.05 mg / L, thiamine hydrochloride 0.01 mg / L, ethylenediamine-N, N, N ', N'-tetraacetic acid disodium salt dihydrate 3.73 mg / L, ferrous sulfate heptahydrate 2.78 mg / L ( pH 5.8).

  The composition of 1/10 MS-Ca medium is as follows: potassium nitrate 190 mg / L, ammonium nitrate 165 mg / L, potassium dihydrogen phosphate 17 mg / L, magnesium sulfate heptahydrate 37 mg / L, boric acid 0.62 mg / L , Manganese sulfate monohydrate 1.69mg / L, Zinc sulfate heptahydrate 0.86mg / L, Sodium molybdate dihydrate 0.025mg / L, Copper sulfate pentahydrate 0.0025mg / L, Cobalt chloride hexahydrate Japanese 0.0025mg / L, potassium iodide 0.083mg / L, myo-inositol 10mg / L, glycine 0.2mg / L, nicotinic acid 0.05mg / L, pyridoxine hydrochloride 0.05mg / L, thiamine hydrochloride 0.01mg / L, ethylenediamine- N, N, N ′, N′-tetraacetic acid disodium salt dihydrate 3.73 mg / L, ferrous sulfate heptahydrate 2.78 mg / L (pH 5.8).

  The 1/10 MS-Ca medium in which calcium ions were removed from the 1/10 MS medium was prepared by preparing a medium without adding calcium chloride dihydrate in inorganic salts.

  The composition of 1/10 MS + EGTA medium is as follows: potassium nitrate 190 mg / L, ammonium nitrate 165 mg / L, potassium dihydrogen phosphate 17 mg / L, magnesium sulfate heptahydrate 37 mg / L, calcium chloride dihydrate 44 mg / L, boric acid 0.62mg / L, manganese sulfate monohydrate 1.69mg / L, zinc sulfate heptahydrate 0.86mg / L, sodium molybdate dihydrate 0.025mg / L, copper sulfate pentahydrate 0.0025 mg / L, cobalt chloride hexahydrate 0.0025 mg / L, potassium iodide 0.083 mg / L, myo-inositol 10 mg / L, glycine 0.2 mg / L, nicotinic acid 0.05 mg / L, pyridoxine hydrochloride 0.05 mg / L, Thiamine hydrochloride 0.01mg / L, ethylenediamine-N, N, N ', N'-tetraacetic acid disodium salt dihydrate 3.73mg / L, ferrous sulfate heptahydrate 2.78mg / L, EGTA 10mM (pH 5.8).

  The composition of 1/10 MS-Ca + EGTA medium is as follows: potassium nitrate 190 mg / L, ammonium nitrate 165 mg / L, potassium dihydrogen phosphate 17 mg / L, magnesium sulfate heptahydrate 37 mg / L, boric acid 0.62 mg / L, manganese sulfate monohydrate 1.69mg / L, zinc sulfate heptahydrate 0.86mg / L, sodium molybdate dihydrate 0.025mg / L, copper sulfate pentahydrate 0.0025mg / L, cobalt chloride hexa Hydrate 0.0025mg / L, potassium iodide 0.083mg / L, myo-inositol 10mg / L, glycine 0.2mg / L, nicotinic acid 0.05mg / L, pyridoxine hydrochloride 0.05mg / L, thiamine hydrochloride 0.01mg / L, ethylenediamine -N, N, N ', N'-tetraacetic acid disodium salt dihydrate 3.73 mg / L, ferrous sulfate heptahydrate 2.78 mg / L, EGTA 10 mM (pH 5.8).

  A GUS assay was then performed as a histochemical analysis. First, extraction buffer (50 mM sodium phosphate buffer (pH 7.0) + 10 mM ethylenediamine-N, N, N ', N'-tetraacetic acid disodium salt dihydrate + 0.1% Triton X-100 + 0.1% lauroyl sarcosine The culture stem section after co-culture with Agrobacterium was immersed in (acid sodium + 10 mM 2-mercaptoethanol) and incubated at 37 ° C. for 1 hour. Then, the solution was exchanged with the above extraction buffer to which the chromogenic substrate stock solution X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) was added to a final concentration of about 1 mM, and the solution was incubated at 37 ° C Incubated further for 1-2 days. Then, in order to decolorize chlorophyll, the culture stem section was treated with 70% ethanol for decolorization. As a result of this GUS assay, blue staining was shown when co-culturing in 1/10 MS + EGTA medium in La France and Bartlett of pear, and in 1/10 MS-Ca + EGTA medium in Japanese pear Sankichi. It was. It was shown that the efficiency of Agrobacterium infection was significantly increased by the addition of co-cultured EGTA.

3. Effects of various treatments on Agrobacterium infection efficiency of cotyledons EGTA (ethylene glycol bis (β-aminoethyl ether) -N, N, N ', N'- for five Agrobacterium cultivars on Agrobacterium infection efficiency The effect of addition of tetraacetic acid to the co-culture medium and the effect of sonication (SAAT) during Agrobacterium infection treatment were examined as follows. A 1/10 MS medium or a 1/10 MS-Ca medium was used as a co-culture medium, and a test group with 10 mM EGTA added and a test group without addition were provided.

  First, prepare cotyledons for five cultivars of Japanese pear (Pyrus pyrifolia (Burm.) Nakai.) (Ninomiya, Natsushizuku, Kosui, Hakuryu, Anshita Shonanashi; went. Ripe seeds of various varieties were surface sterilized with 0.1% hydroxyquinoline and stored at 4 ° C.

  The mature seeds were treated with 80% ethanol for 30 seconds and then sterilized by treatment with a sodium hypochlorite solution (approximately 1% effective chlorine) added with a few drops of Tween 20 for 20 minutes. The seeds were then rinsed twice with sterile water.

  After that, the seed coat, hypocotyl, and 1/3 of the cotyledon that are close to the hypocotyl are removed from the seed, and the two cotyledons are divided into one, and one cotyledon is further divided into two along the main vein. Was one explant. The obtained explants were immersed in a cutting medium (1/10 MS-Ca + 6.3% sucrose + 3.6% glucose + 10 mM MES (2-morpholinoethanesulfonic acid) (pH 5.8)).

  The explant (part of cotyledon) taken out from the cutting medium was immersed in the Agrobacterium suspension prepared above. Some explants were subjected to ultrasonic treatment (SAAT treatment) at this stage. Sonication was performed for 20 seconds using an iuchi ultrasonic cleaner US-2 (oscillation frequency 38 KHz). Subsequently, Agrobacterium infection treatment was carried out by culturing in an Agrobacterium suspension (GFP / LBA4404 strain) for 15 minutes.

  Remove the explants from the Agrobacterium suspension and blot the excess bacteria with sterile filter paper, then add 3% sucrose + 10 mM MES + 100 μM acetosyringone + 5 μM NAA + 10 μM BA (or 10 μM TDZ) + 0.85% agar. Transfer to the added 1/10 MS medium, 1/10 MS-Ca medium, 1/10 MS + EGTA medium, or 1/10 MS-Ca + EGTA (both pH 5.8) medium, in the dark at 26 ° C For 5 days.

  After co-culture, the explants were washed with an MS liquid medium containing 400 μg / ml claphoran. Thereafter, the explants were cultured in a regeneration medium (MS medium + 5 μM NAA + 10 μM BA (or 10 μM TDZ) +5 μg / ml kanamycin + 200 μg / ml claphoran + 0.85% agar) at 26 ° C. under dark conditions, Selection and redifferentiation were induced. Once a month, the cells were subcultured to a new regeneration medium having the same composition.

  About the obtained redifferentiated individual, GFP fluorescence was observed using the Leica MZ FLIII stereofluorescence microscope (480/40 nm excitation filter, 50 nm barrier filter) of the redifferentiated individual.

  Redifferentiated individuals (redifferentiated plants, adventitious shoots, adventitious roots, and tissues derived from planted tissues) that have been observed GFP fluorescence 2 weeks after the start of co-cultivation using 1/10 MS-Ca medium as the coexisting medium Table 1 shows the result of comparing the ratio of each process.

  As shown in Table 1, GFP fluorescence was observed in 34% of the test group in which neither EGTA addition nor sonication (SAAT) was performed, and 44% in the test group in which only EGTA was added. In addition, GFP fluorescence was observed in 64% of the redifferentiated individuals in the test group where only sonication (SAAT) was performed, and 70% of the redifferentiated individuals in the test group where both EGTA addition and sonication (SAAT) were performed. GFP fluorescence was observed. Here, the rate at which GFP fluorescence was observed indicates the rate at which Agrobacterium was infected and transformation was established or the transgene was expressed in the cell. Agrobacterium infection efficiency is significantly increased by adding EGTA and co-cultivating plant tissue fragments and Agrobacterium in a plant tissue culture medium excluding calcium ions and applying sonication (SAAT). It was shown that transformants can be produced.

  Furthermore, for comparison, the ratio of GFP fluorescence was also examined for redifferentiated individuals 2 weeks after the start of co-culture when 1/10 MS medium was used as the co-culture medium. As an example, Table 2 shows the results for Anshita Shonanashi.

  As shown in Table 2, GFP fluorescence was observed in 3% in the test group where neither EGTA addition nor sonication (SAAT) was performed, and 10% in the test group where only EGTA addition was performed. In addition, GFP fluorescence was observed in 22% of the redifferentiated individuals in the test group where only sonication (SAAT) was performed, and 36% of the redifferentiated individuals in the test group where both EGTA addition and sonication (SAAT) were performed. GFP fluorescence was observed. These values were clearly lower than when 1/10 MS-Ca medium was used (Table 1).

  On the other hand, the proportion of redifferentiated individuals in which GFP fluorescence was observed about 5 months after the start of infection using 1/10 MS-Ca medium as the co-culture medium was as shown in Table 3.

  As shown in Table 3, GFP fluorescence was observed in 17% of the re-differentiated individuals in the test group where neither EGTA addition nor sonication (SAAT) was performed, and in the test group where only EGTA addition was performed. In addition, GFP fluorescence was observed in 43% of the redifferentiated individuals in the test group where only sonication (SAAT) was performed, and 47% of the redifferentiated individuals in the test group where both EGTA addition and sonication (SAAT) were performed. GFP fluorescence was observed. Plant parts such as explants are sonicated (SAAT) in Agrobacterium solution and co-cultured with Agrobacterium in a co-culture medium containing EGTA and excluding calcium ions Thus, it was shown that the efficiency of Agrobacterium infection can be remarkably increased, and that the transformant can be maintained at a relatively high rate over a long period of 5 months or longer.

  The results are roughly categorized by the presence or absence of EGTA and the presence or absence of SAAT treatment. The redifferentiated individuals showing GFP fluorescence after 5 months of coculture were 122/324 (37.7%) in the EGTA-added test group and no EGTA. It was 39/122 (32.0%) in the additive test group, 109/238 (45.8%) in the SAAT-treated test group, and 52/208 (25.0%) in the SAAT-untreated test group. The effect of increasing Agrobacterium infection efficiency by adding EGTA and sonication was shown.

4). Agrobacterium infection efficiency of cotyledons Redifferentiated individuals (adventitious buds) exhibiting GFP fluorescence as described above were cultured in a medium (MS medium + 0.1 μM NAA + 2.5-10 μM BA + 0.85% agar (pH 5.8) +5 μg / ml kanamycin + 200 μg / In order to promote the growth to plants, the cells were cultured for 16 hours in the light period and 8 hours in the dark.

  GFP fluorescence from explants cultured on 1/10 MS-Ca medium + 10 mM EGTA + 3% sucrose + 10 mM MES + 100 μM acetosyringone + 5 μM NAA + 10 μM BA + 0.85% agar (pH 5.8) after SAAT treatment One adventitious bud of a Japanese pear (variety: Anshita Shonanashi) that showed the above was obtained (FIG. 2). In this adventitious bud individual, GFP fluorescence was detected throughout each developmental stage (FIGS. 2A-C).

  Genomic DNA was extracted from the cultured leaves using DNeasy plant mini kit (Qiagen). As a control, genomic DNA was extracted from leaves of non-transformed plants grown in the field using QIAGEN Genomic-tip (Qiagen). The extraction method followed the manual. Using the obtained genomic DNA as a template, the integration of the nucleic acid construct (T-DNA) containing the GFP gene into the Japanese pear genome was confirmed by PCR.

  For PCR, as primer set A, the primer designed in the 35S promoter region (5'-GATGTGATATCTCCACTGACGTAAG-3 '; SEQ ID NO: 1) and the primer designed in the NOS terminator (5'-GTATAATTGCGGGACTCTAAT-3' SEQ ID NO: 2) was used. PCR cycle conditions were 95 ° C for 5 minutes in 1 cycle, 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1.5 minutes for 30 cycles, and 72 ° C for 7 minutes in 1 cycle. By this PCR, the 35S-gfp-Nos gene part (about 1 kb) was amplified. Furthermore, using primer set B (5'-AGCTGACCCTGAAGTTCATC-3 '(SEQ ID NO: 3) and 5'-GTGTTCTGCTGGTAGTGGTC-3' (SEQ ID NO: 4)) designed in the GFP gene, 1 cycle at 94 ° C for 5 minutes The GFP gene portion (about 400 bp) sandwiched between both primers was amplified by PCR in 35 cycles of 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 2 minutes, and 72 ° C for 7 minutes in 1 cycle. From the transformant-derived genomic DNA, an amplification product of the desired size (approximately 1.0 kb) was obtained with the primer set in the 35S promoter region and the primer in the NOS terminator designed to sandwich the GFP gene. Was shown (FIG. 3A). Moreover, it was shown that the amplification product of the target size (about 0.4 kb) was obtained from the transformant-derived genomic DNA by the primer set designed in the GFP gene (FIG. 3B).

  Furthermore, genomic Southern analysis was performed using the DIG DNA Labeling Kit (Roche) and a probe designed in the GFP gene. First, genomic DNA was digested with DraI and transferred to a nylon membrane (Hybond-N, Amersham). As a probe, an amplification product (about 400 bp) obtained by using promoters 5′-AGCTGACCCTGAAGTTCATC-3 ′ (SEQ ID NO: 3) and 5′-GTGTTCTGCTGGTAGTGGTC-3 ′ (SEQ ID NO: 4) designed in the GFP gene was used as DIG -Labeled with PCR was used. In the genomic Southern analysis, the detection result of the genomic DNA fragment with the probe of the GFP gene fragment confirmed the introduction of 3 copies of the GFP gene into the genomic DNA in the Japanese pear transformant (FIG. 4).

  From the above results, it was confirmed that the obtained adventitious bud was a transformant in which a nucleic acid construct containing a GFP gene in the genome was incorporated. The adventitious shoots could be grown on a medium of MS medium + 0.1 μM NAA + 2.5-10 μM BA + 0.85% agar (pH 5.8), and could be potted by bud-contacting.

  Therefore, it has been shown that the use of the method of the present invention makes it possible to produce a stable transformant in which the target gene of Japanese pear is integrated into the genome.

  The method of the present invention can be used to produce a stable transformant of Japanese pear. By using the present invention, a pest-resistant rootstock introduced with genes that are resistant to diseases such as herringbone disease and nematodes, and genes that are resistant to drought and salt stress were introduced into existing Japanese pear species. It becomes possible to produce stress-resistant rootstocks, and to provide a solution for the problems occurring in Japanese pear cultivation. Recently, gene analysis related to the target phenomenon (character) in Japanese pear has progressed by microarray analysis. By using the present invention, emphasis on “flower bud formation”, “dormant”, “mitschism”, etc. It is also possible to elucidate the functions of genes necessary to solve research issues, and further research is expected to develop new cultivation techniques adapted to global warming.

  Sequence number 1-4: It is a primer.

Claims (4)

  A method for transforming Japanese pears using the Agrobacterium method, wherein the plant cells or plant body parts derived from Japanese pears are sonicated in the presence of Agrobacterium holding T-DNA containing the transgene, and the plant A method comprising coculturing a cell or a plant part with the Agrobacterium using a coexisting medium containing a calcium ion chelator and excluding calcium ions.   The method according to claim 1, wherein the co-culture medium is prepared using an MS medium or an MS medium having a diluted inorganic salt concentration as a basic medium.   The method according to claim 1 or 2, wherein the calcium ion chelating agent is ethylene glycol bis (β-aminoethyl ether) -N, N, N ', N'-tetraacetic acid.   The method according to claim 1, wherein the plant cell or plant part is derived from cotyledons.

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