JP2010227033A - Method for producing plant transformant and plant transformant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a plant transformant using a tissue or organ of a plant of the genus Coptis as a material; and to provide a plant transformant. <P>SOLUTION: The method for producing a plant transformant includes: a sterilization step for sterilizing a tissue or organ of a plant of the genus Coptis; a plant tissue culture induction step for inducing a plant tissue culture from a sterilized tissue or organ; a subculture step for subculturing the plant tissue culture; a callus induction step for inducing a callus from the induced or subcultured tissue culture; and a transformation step for transferring a gene to the callus and transforming the callus. In the subculture step and the callus induction step, the culture is aseptically cultured and in the transformation step, the culture is redifferentiated through a callus into a plant tissue or organ. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing a plant transformant using a tissue / organ of a plant belonging to the genus Oren, and a plant transformant.

  The plant of the genus Auricularis is a perennial plant of the Ranunculaceae family, and exhibits various pharmacological activities such as antibacterial and antiviral effects, blood pressure lowering effects, anti-inflammatory effects, antispasmodic effects, analgesic effects, and bile secretion promoting effects. Also produces important berberine.

  Among the plants of the genus Coptis (genus Coptis), in particular, our plant (Coptis japonica Makino), Coptis chinensis Franchet, Coptis dertoidea sea chen Chen et. The rhizomes of Coptis deltoidea CY Cheng et Hsiao and Coptis teeta Wallichi are drugs listed in the 15th revision of the Japanese Pharmacopoeia as the crude drug “Ouren” Patent Document 1).

  Herbal medicine “Yelen” is incorporated into gastrointestinal drugs as antidiarrheal and healthy stomach medicine, and is also incorporated into Kampo prescriptions for the treatment of hot flashes, mental anxiety and hyperemia. One. More recently, an action to lower blood cholesterol has been reported as a new pharmacological action of berberine (see Non-Patent Document 2). For this reason, the plant of the genus Auren is considered promising as a therapeutic agent for metabolic syndrome, and the demand is expected to increase in the future.

  Traditionally, auren has been cultivated in various parts of Japan such as Hyogo, Fukui, Tottori and Ishikawa. However, it takes 5 to 10 years to harvest the plant, it takes time to prepare herbal medicines, the aging of rural areas, and the increase in cheap imported products from China. In 2001, domestic production was about 6 tons compared to 100 tons of imports in 2001. Furthermore, in 2007, only 1.8 tons were produced in three prefectures, Shizuoka Prefecture, Toyama Prefecture, and Fukui Prefecture (see Non-Patent Document 3).

  In order to revitalize the domestic production of genus Aurora, it is desirable to cultivate varieties that can yield high yields of berberine in short-term cultivation, and can be cultivated in closed greenhouses that are not affected by weather and natural disasters Breeding various varieties is also effective. For this reason, the use of genetic recombination methods in order to achieve the breeding of varieties that previously required a long time in a short period of time is particularly effective for plant species with slow growth, such as the genus Oren. . A genetic recombination method is a method of introducing a new trait or strengthening or suppressing a specific trait by introducing a gene of self or another organism having a specific function into the genome chromosome of the target organism. .

  As for the genetic recombination of the plant belonging to the genus Aureen, there are reports of transformation of cultured cell of auren and transformation using a petiole of a plant cultivated outdoors as an explant (Non-patent Documents 4 and 5). reference).

  According to the method disclosed in Non-Patent Document 4, the transformation of cultured aurene cells is only 1 clone from 16 g (160 million) cultured cells, and the transformation efficiency is high. It was very low and practical.

  In the method disclosed in Non-Patent Document 5, transformed cells were formed in 33.3% of the explants, and the transformation efficiency was dramatically improved. From early summer to early spring, when it was difficult to obtain fresh plant material, transformation experiments could not be performed.

  For this reason, a new production method suitable for producing a plant transformant having high transformation efficiency without being affected by the time when a plant belonging to the genus Oren can be collected is required.

Fifteenth revision Japanese Pharmacopoeia, 1187, 2006 W. Kong et al., Nat. Med. 10, 1344-1351 (2004) Documents related to medicinal crops (herbal medicine), March 2007, Japan Special Agricultural Products Association, p16 (2007) F. Sato et al., PNAS 98, 367-362 (2001) N. Shitan et al., Plant Biotechnology 22, 113-118 (2005)

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a method for producing a plant transformant using a tissue / organ of a plant belonging to the genus Oren and a plant transformant.

In order to achieve the above object, a method for producing a plant transformant according to the first aspect of the present invention comprises:
A sterilization process for sterilizing tissues and organs of the genus Oren,
Plant tissue culture induction step of culturing the sterilized tissue / organ and inducing a plant tissue culture from the tissue / organ;
A subculturing step of subculturing the induced plant tissue culture;
A callus induction step of inducing callus from the induced or subcultured plant tissue culture;
And a transformation step of transforming the callus by introducing a gene into the callus derived from the plant tissue culture.

In the subculture step, the plant tissue culture is aseptically cultured,
In the transformation step, the callus can be redifferentiated into a plant tissue or organ.

  In the subculture process and the callus induction process, the culture can be performed in a solid medium containing auxin and cytokinin.

  In the transformation step, the gene can be introduced by at least one of the Agrobacterium method, the polyethylene glycol method, the electroporation method, and the particle gun method.

  In addition, a plant transformant according to another aspect of the present invention is characterized by being transformed by introducing a norcoclaurine-6-O-methyltransferase (6-OMT) gene.

  ADVANTAGE OF THE INVENTION According to this invention, the plant transformant which used the structure | tissue / organ of the genus Orenum as a material can be produced. Moreover, the plant transformant of the plant of the genus Oren can be produced with high efficiency.

It is a schematic diagram which shows the structure of binary vector pBI-sGFP. It is a figure which shows the fluorescence of sGFP which appeared as a result of performing sGFP gene introduction | transduction to the auricular callus which concerns on Example 1. FIG. It is a figure which shows the transformed callus clone obtained by isolate | separating and subculturing a part of callus in which sGFP fluorescence which concerns on Example 1 was observed. It is a figure which shows the sGFP fluorescence of the transformed callus clone obtained by isolate | separating and subculturing a part of callus in which sGFP fluorescence which concerns on Example 1 was observed. It is a figure which shows the fluorescence of the sGFP which emerged as a result of performing sGFP gene introduction | transduction to the petal of the auren cultured plant body and cultured shoot which concern on Example 2. FIG. It is a figure which shows the fluorescence of the sGFP which appeared as a result of performing sGFP gene introduction | transduction to the cultured root of the plant of the genus Orenum concerning Example 3. It is a figure which shows the callus transformed by introducing the 6-OMT gene according to Example 4. It is a figure which shows the callus transformed by introducing the 6-OMT gene according to Example 4. It is a figure which shows the 6-OMT gene introduction | transduction transformed plant body redifferentiated from the callus transformed by introducing the 6-OMT gene which concerns on Example 4. It is a figure which shows that 6-OMT gene was introduce | transduced into the Aurenium plant. It is a figure which shows that 6-OMT gene was introduce | transduced into the Aurenium plant.

  Hereinafter, the present invention will be described in detail. Throughout this specification, it should be understood that expressions in the singular system also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

In the present invention, “plant tissue culture” refers to a product obtained by aseptically separating various organs, tissues or cells of a plant and culturing them aseptically under appropriate conditions. Specifically, callus, somatic embryo, cultured shoot, cultured root, cultured plant and the like.
The “cultivated plant body” refers to a plant tissue culture having a plant morphological aboveground tissue “stems and leaves” and an underground tissue “root”.
“Culture shoot” refers to a plant tissue culture having a plant morphological above-ground tissue “stems and leaves” and having no “root”.
“Culture root” refers to a plant tissue culture having a plant morphological underground tissue “root” and having no “stems and leaves”.
“Callus” refers to a plant cell culture that is an abundant mass of plant cells that does not have plant morphological ground tissue “stems and leaves” or underground tissue “root”.
“Somatic embryo” is an “embryoid body” of plant tissue culture derived from somatic cells of a plant individual, and has the same functions and morphogenesis as normal seed embryos derived from fertilized eggs Say.

  The plant which is the subject of the present invention is not particularly limited as long as it is a plant included in the genus Coptis (hereinafter, genus Cyprus). Examples of the plant belonging to the genus Coptis, for example, Coptis japonica, Coptis chinensis, Coptis deltoidea, and other genus Coptis plants whose main alkaloid is berberine. Can be used.

  In the present invention, the term “sterile plant” refers to a product obtained by sterilizing various organs, tissues or seeds of a plant cultivated in the field in addition to the plant tissue culture and cultivating aseptically under appropriate conditions. Also say. Aseptic plants are cultivated in an aseptic space by putting the medium necessary for plant growth into a sealed container such as a test tube or flask, sterilizing both the medium and the container using an autoclave, etc. .

In the present invention, “auxin” refers to indoleacetic acid (IAA) having the property of causing cell elongation, rooting, cell division promotion, callus formation, etc., among “plant growth regulators” that promote plant cell growth. ), Indolebutyric acid (IBA), naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and the like.
“Cytokinein” is a “plant growth regulator” of isopentenyl adenine (IPA), kinetin (KIN), zeatin, and benzyladenine that promotes cell division, adventitious bud formation, bud growth, etc. BA).

  A “plant expression vector” is an appropriate vector containing a desired recombinant gene in order to introduce the desired recombinant gene into a plant. Plant expression vectors can be prepared using gene recombination techniques well known to those skilled in the art. For the construction of a plant expression vector for use in a transformation method using Agrobacterium, for example, a pBI vector is preferably used, but the present invention is not limited thereto.

A “desired recombinant gene” refers to any polynucleotide that is desired to be introduced into a plant. The desired recombinant gene in the present invention is not limited to those isolated from nature, and may include synthetic polynucleotides. A synthetic polynucleotide can be obtained, for example, by synthesizing or modifying a gene having a known sequence by a technique well known to those skilled in the art.
The desired recombinant gene in the present invention includes, for example, any polynucleotide that is endogenous or exogenous to the plant, whose expression is desired in the transformed plant, and the expression of the endogenous gene in the plant. A polynucleotide containing an antisense sequence of its target gene when control is desired. Also, alkaloid biosynthesis key enzyme gene, alkaloid biosynthesis enzyme gene inhibitory sequence (RNAi), transcription factor affecting RNA synthesis ability of alkaloid and its RNAi, plant morphology (for example, leaf growth, rapid growth A transcription factor derived from Arabidopsis thaliana and its repressing gene that affect the plant height, and a homologous gene derived from Ceribauren, a transcription factor that affects the morphology of plants.

  Where expression is intended in plants, the desired recombinant gene comprises a self promoter, i.e. a promoter to which the gene is operably linked in nature, or comprises a self promoter. If not, or if it is desired to further include a promoter other than its own promoter, it is operably linked to any suitable plant promoter. Plant promoters that can be used include constitutive promoters and promoters that are selectively expressed in parts of the plant, as well as inducible promoters.

  In the plant expression vector, various elements can be further linked in a state where they can operate in the cells of the host plant. The regulatory elements can suitably include a selectable marker gene, a plant promoter, a terminator and an enhancer. It is well known to those skilled in the art that the type of plant expression vector used and the type of regulatory element can vary depending on the purpose of transformation.

  A “selection marker gene” can be used to facilitate selection of transformed plants. Hygromycin phosphotransferase (hpt) gene for conferring hygromycin resistance, neomycin phosphotransferase II (nptII) gene for conferring kanamycin resistance, and phosphinothricin acetyltransferase for conferring bialaphos resistance A drug resistance gene such as (pat) gene can be preferably used, but is not limited thereto.

  The “reporter gene” is usually a fluorescent protein gene or a gene of an enzyme that catalyzes a color reaction. For example, a green fluorescent protein (GFP) gene, a red fluorescent protein gene (DsRed), a luciferase gene (Luc), or β -Glucuronidase (GUS) gene. The reporter gene can be used to clarify the aspect of plant tissue / cell-specific expression of a recombinant gene introduced into a transformed plant, and a selection marker for facilitating the selection of transformed plants It can also be used as a gene. As a selection marker gene other than the drug resistance gene, a green fluorescent protein (GFP) gene or a red fluorescent protein gene (DsRed) can be preferably used, but is not limited thereto.

  “Plant promoter” means a plant-expressed promoter operably linked to a selectable marker gene and a desired recombinant gene. Examples of such promoters include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter and the promoter of nopaline synthase.

  A “terminator” is a sequence that is located downstream of a region encoding a protein of a gene, and is involved in termination of transcription when DNA is transcribed into mRNA, and addition of a poly A sequence. Examples of the terminator include, but are not limited to, CaMV35S terminator and terminator (Tnos) of nopaline synthase gene.

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. A plurality of enhancers can be used per one plant expression vector.

  Hereinafter, a method for transforming the plant belonging to the genus Orensis will be described.

(Sterilization treatment)
First, the tissues and organs constituting a part of the cultivated Aurelia plant are sterilized. The tissue / organ is, for example, a leaf, a petiole, an inflorescence, a stem, a rhizome, a root, a fruit, or a seed. In order to sterilize cultivated genus plant in the field, (1) sterilize with 75% ethanol for 1 minute, (2) rinse with sterilized water for 1 minute, (3) 2% sodium hypochlorite (1 drop / Tween20 / 30 ml) for 10 minutes, (4) rinse with sterile water for 1 minute three times, and (5) remove the damaged part of the plant.
Any known sterilization method that can reduce the number of bacteria attached to the plant to a certain number or less can be used.

  Although the above-mentioned treatment can sterilize cultivated genus plant of the outdoors, etc., since it is a plant cultivated in the field, it is difficult to obtain a sterile plant suitable for transformation. For this reason, an aseptic plant can be produced by performing the plant tissue culture induction process and the subculture process described below.

(Plant tissue culture induction treatment)
Next, sterilized Auren plant tissues / organs are prepared into sections of an appropriate size (for example, 5 mm in length), and then auxin (for example, 0-1 mg / l NAA) at an appropriate concentration and cytokinin are prepared. (For example, 0 to 2 mg / l KIN). As a result of the treatment, a sterile plant can be produced.

  As the medium at this time, Murashige-Skoog medium, Gambolg B5 medium, White medium, Linsmeier-Skoog medium, Woody plant (WP) medium (G. Lloyd & B. McCown, Intnl. Plant Propag. Soc. Combd. Procd. , vol. 30, 421-427 (1980)) or the like can be used. In particular, it is desirable to use a WP medium, and it is further desirable to add 10 mg / l L-glutamine to this medium. The culture temperature is typically 15 to 25 ° C, preferably 20 ° C. The light conditions are not particularly limited, but a dark place is desirable.

(Subculture treatment)
By performing at least one culture, a plant tissue culture is produced. Then, the produced plant tissue culture is subcultured as it is or by dividing and replanting. The number of subcultures is arbitrary, and it is sufficient that a plant tissue culture can be produced. In the subculture treatment, a medium similar to the plant tissue culture induction treatment can be used.

  In addition, the plant tissue culture produced by one culture without subculture can also be subjected to the transformation treatment described later, and the plant tissue culture produced by subculture is transformed. You can also

(Transformation treatment)
Next, the cultured plant tissue culture is transformed. The Agrobacterium method will be described as a method for transformation.

The Agrobacterium used for the transformation of the plant of the genus Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes, preferably Agrobacterium rhizogenes. is there. Agrobacterium is a plant expression vector containing a desired recombinant gene and is transformed, for example, by electroporation (electroporation). A desired recombinant gene can be introduced into a plant by infecting the plant tissue culture of the plant belonging to the genus Aurelia with the transformed Agrobacterium. The introduced recombinant gene is integrated in the genome of the plant. In addition, a selection marker gene for selecting a transformant and a non-transformant is introduced into a plant together with a desired recombinant gene.
The plant genome includes not only nuclear chromosomes but also genomes contained in various organelles such as mitochondria and chloroplasts in plant cells.

(Callus induction treatment and Agrobacterium infection treatment)
The plant tissue culture (for example, callus, somatic embryo, cultured shoot and root) has an appropriate concentration of auxin (for example, 0-1 mg / l NAA) and cytokinin (for example, 0-2 mg / l KIN). After the callus induction treatment in a medium containing the above, it is prepared to an appropriate size (for example, a cell mass having a length of 5 mm and a diameter of 2 to 3 mm) and infected with transformed Agrobacterium under aseptic operation.

  During infection with Agrobacterium (co-culture), plant sections are typically kept warm in the dark for 30 minutes to 14 days, preferably 14 days. The general period for co-culturing plants and Agrobacterium is about 2 to 3 days, but the co-cultivation period in this transformation treatment is preferably longer than the general co-culture period.

  The temperature during co-culture is typically 10 to 28 ° C, preferably 20 ° C. By reducing the temperature during co-cultivation, the amount of berberine produced from the plant belonging to the genus Oren can be suppressed. Berberine has a bactericidal effect, and if the amount of berberine is suppressed, the bactericidal effect is weakened, so that the transformation efficiency can be increased.

  Next, in order to sterilize Agrobacterium, the plant section co-cultured is treated with an appropriate sterilizing agent (for example, carbenicillin, kraforan).

  A transformed plant cell is selected based on a selection marker (for example, drug resistance such as kanamycin resistance and hygromycin resistance) contained in the plant expression vector.

  Agrobacterium sterilization treatment and selection of transformed cells using a selection marker can be performed simultaneously by adding both agents to the medium, but each can also be performed independently. In particular, in sterilization treatment immediately after Agrobacterium infection, it is preferable to add a sterilizing agent alone without adding a drug for selection.

  After culturing under appropriate sterilization conditions and selection conditions, the selected transformed cells are redifferentiation medium containing an appropriate plant growth regulator (eg, WP medium containing 10 mg / l glutamine, hereinafter referred to as WPG medium). And kept warm for an appropriate period. The redifferentiated transformant is transferred to a rooting medium (for example, a WPG medium not containing a plant regulator). After root development is confirmed, the transformants are raised.

  The desired recombinant gene introduced into the plant acts for the intended purpose in the plant (eg, the expression of the desired new trait or the control of the expression of some endogenous gene).

Whether or not the desired recombinant gene has been introduced into the plant can be confirmed using techniques well known to those skilled in the art. This confirmation can be performed by, for example, PCR method, Southern hybridization method, Northern hybridization method, in situ hybridization and the like. Specifically, in the case of the PCR method, DNA is prepared from a transformed plant, and PCR is carried out by designing a desired recombinant gene-specific primer. After PCR, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band. By doing so, it can confirm that it transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, the amplification product may be bound to a solid phase such as a microplate, and the amplification product may be confirmed by fluorescence or enzymatic reaction.
When a reporter gene is introduced into a plant as a recombinant gene, the introduced gene can be confirmed without preparing DNA or RNA from the transformed plant. For example, when a modified GFP (sGFP) gene is introduced into a plant, the recombinant gene is introduced and the expressed plant cells produce sGFP protein, and thus specific green fluorescence is observed. That is, gene transfer can be confirmed by observation under a fluorescence microscope.

  The transformation efficiency can be calculated by dividing the number of plant sections into which the desired recombinant gene has been inserted into the genomic DNA by the number of sections subjected to infection and multiplying by 100. When the sGFP gene is used as a recombinant gene to be introduced, callus formed on a plant section that has been selected by Agrobacterium infection, sterilization, and drug resistance marker is observed under a fluorescence microscope, and sGFP fluorescence is observed. The number of sections obtained is divided by the number of sections subjected to infection and multiplied by 100. If sGFP fluorescence is observed in any part of the callus formed in one section, it is counted as 1. For sGFP fluorescence, the non-recombinant callus is observed before observing the test section, and the test section is observed after setting the excitation light and exposure conditions such that no fluorescence is observed in the non-recombinant callus.

  The plant transformant produced by the method according to the present invention has a transformation efficiency of 27 to 70%, which is about twice or more the efficiency of the conventional method.

  The method for transforming the plant tissue culture is not limited to the Agrobacterium method, and any known method such as a polyethylene glycol method, an electroporation method, or a particle gun method can be used. In addition, the transformation treatment can also be performed using a plurality of methods, for example, two methods, an Agrobacterium method and a polyethylene glycol method.

  As described above, according to the present invention, it is possible to produce a plant transformant using a tissue / organ of a plant belonging to the genus Oren. Moreover, the plant transformant of the plant of the genus Oren can be produced with high efficiency.

  In the conventional method, a petiole explant of a field-cultivated plant is used as a material for transformation. Therefore, sterilization of the petiole is required before infection with Agrobacterium. In the season when the plant of the genus Aurensis cannot be collected, the material for transformation itself is not available, so the season in which transformation is possible was limited from early spring to early summer. In contrast, the method of the present invention uses a sterilized plant tissue culture, which allows for year-round transformation.

  Next, a method for producing a transformant of a plant belonging to the genus Oren that is transformed by introducing a norcoclaurine-6-O-methyltransferase (6-OMT) gene will be described. In addition, about the process which disinfects a genus plant, the process which induces a plant tissue culture, and the process which transforms, it is the same as that of the above-mentioned process.

(vector)
In order to introduce a desired recombinant gene into a plant belonging to the genus Auren, an appropriate plant expression vector containing the desired recombinant gene is constructed. A cassette in which a 6-OMT gene is linked downstream of a plant high expression promoter (EL2-35S), a kanamycin resistance gene (nptII) or a hygromycin resistance gene (hpt), which is a marker gene for selecting transformed cells, A binary vector having T on DNA is used.

  Whether 6-OMT gene has been introduced is confirmed by extracting DNA from callus or transformed plant body using DNeasy (QIAGEN) according to the conventional method, and expressing the high expression promoter (EL2-35S) and 6-OMT gene. It is performed by PCR using a specific primer set. Then, the sequence from the introduced EL2-35S to the 6-OMT gene is confirmed by electrophoresis. The transformation can also be confirmed by a PCR method using a primer set specific for each of the marker genes kanamycin resistance gene (nptII) or hygromycin resistance gene (hpt).

  The 6-OMT gene is an isoquinoline alkaloid biosynthesis gene. Examples of isoquinoline alkaloid biosynthetic genes other than 6-OMT genes include tyrosine decarboxylase (TYDC), norcoclaurine synthase (NCS), coclaurin-N-methyltransferase (CNMT), cytochrome P450 80B2 (CYP80B2), ( S) -3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase (4'OMT), berberine bridge enzyme (BBE), squalelin-9-O-methyltransferase (SMT), or cytochrome P450 719A: A gene encoding canadine synthase (CYP719A) and the like can be mentioned. By introducing an isoquinoline alkaloid biosynthetic gene other than the 6-OMT gene, a plant of the genus Oren can be transformed.

  As described above, according to the present invention, a plant transformant of the genus Auren into which a 6-OMT gene is introduced can be produced.

  The present invention will be specifically described below with reference to examples. The present invention is not limited to the following examples. The materials, reagents, etc. used in the examples are available from commercial sources unless specified otherwise.

(Example 1: Transformation of callus)
Using callus of a plant belonging to the genus Auren (Coptis japonica Makino), transformation was carried out by the following method.

(vector)
FIG. 1 is a schematic diagram showing the structure of the binary vector pBI-sGFP. Agrobacterium tumefaciens is a binary vector pBI-sGFP having a kanamycin resistance gene (nptII) which is a marker gene for selecting transformed cells and a sGFP (synthesized Green Fluorescent Protein) gene serving as a reporter for transformed cells on T-DNA. LBA4404 bacteria were transformed and used for experiments.

(Callus induction and callus subculture)
The petioles of the sterilized oren field-cultivated plant were placed on a WPG medium containing 10 mg / l L-glutamine and cultured in the dark at 20 ° C. to induce callus having somatic embryogenic ability. The obtained callus was subcultured in the same medium. The composition of WPG medium (WPGN1K2 medium) used for callus induction and subculture is as follows: 30 g / l sucrose, 10 mg / l L-glutamine, 1 mg / l NAA, 2 mg / l KIN, 2. 5 g / l gellite, pH 5.7.

(Agrobacterium infection)
Agrobacterium in culture in a YEB solid medium at 25 ° C. in the dark was scooped with a platinum loop and suspended in sterile water containing 20 mg / l acetosyringone to prepare an Agrobacterium solution. A callus piece having a diameter of 2-3 mm was prepared from the above callus, immersed in an Agrobacterium solution as it was, and transplanted to a WPGN1K2 medium, and co-cultured in the dark at 20 ° C. for 14 days.

(Selection of sterilized and transformed callus)
After completion of co-cultivation, callus pieces infected with Agrobacterium were placed in sterilized water (Klaforan solution) containing 1 g / l Claforan, shaken at 100 rpm at 20 ° C. for 1 day in the dark, and then Claforan solution. The Agrobacterium was washed away from the callus pieces for 3 days, and the kraforan solution was changed for 4 days, and this operation was repeated 3 times in total for 4 days.

  Subsequently, in order to select transformed cells, the cells were placed on WPGN1K2 medium (selection medium) containing 20 mg / l geneticin as a drug. When Agrobacterium began to grow again from the callus piece, it was transplanted to a WPGN1K2 medium (sterilization medium) supplemented with 500 mg / l Kraforan and sterilized. Moreover, when the browning of the callus piece was remarkable due to the influence of the drug for sterilization and the drug for selection, the callus piece was transplanted to a WPGN1K2 medium containing no drug to promote callus growth. The appearance of transformed cells was evaluated using the reporter gene sGFP expression as an indicator.

  FIG. 2 shows the fluorescence of sGFP that appears as a result of introducing the sGFP gene into the genus Callen. Regarding the fluorescence of sGFP, the callus was irradiated with excitation light (blue light), and the emitted green fluorescence (510 nm) was observed with a stereoscopic fluorescence microscope (OLYMPUS SZX16). As shown in FIG. 2, it was found that the sGFP gene was introduced into callus of the genus Oren and a transformant was obtained from the callus.

  FIG. 3A is a transformed callus clone obtained by isolating and subculturing a part of the callus in which sGFP fluorescence was observed in FIG. Moreover, FIG. 3B is a figure which shows the sGFP fluorescence of the callus clone of FIG. 3A. Regarding the fluorescence of sGFP, the callus was irradiated with excitation light (blue light), and the emitted green fluorescence (510 nm) was observed with a stereoscopic fluorescence microscope (VG-05 series, Keyence Corporation). As described above, it was found that a callus clone showing uniform sGFP fluorescence expression was obtained by separating and cloning the callus obtained by the transformation treatment.

(Example 2: Transformation of cultured plant body and cultured shoot)
Transformation was carried out by the following method using cultured plant bodies and cultured shoots of the plant of the genus Ouren (Coptis japonica Makino).

(vector)
Similar to Example 1, Agrobacterium tumefaciens LBA4404 is a binary vector pBI-sGFP having a kanamycin resistance gene (nptII) which is a marker gene for selecting transformed cells and a sGFP gene serving as a reporter for transformed cells on T-DNA. The fungus was transformed and used for the experiment.

(Callus induction)
A section of about 5 mm length was prepared from the petite of cultured aurene and cultured shoots, planted in a WPGN1K2 solid medium supplemented with 2% sucrose, and cultured at 20 ° C. in the dark for 30 days.

(Agrobacterium infection)
Agrobacterium grown in an LB liquid medium at 28 ° C. in the dark was used as the bacterial solution. The petiole slice in which callus is formed on both ends of the culture is placed in a WPGN1K2 liquid medium containing 2% sucrose, an appropriate amount of Agrobacterium solution is added, and 1 million to 1,000 per 1 ml of the medium is added. It was prepared so that it would become a million Agrobacterium, and infection treatment was performed at 28 ° C. for 30 minutes while shaking at 60 rpm in the dark.

(Selection of sterilized and transformed callus)
After completion of the infection treatment, the petiole slice infected with Agrobacterium was placed on a WPGN1K2 solid medium (sterilization medium) containing 500 mg / l claforan, and sterilization treatment was performed.

  Subsequently, in order to select transformed cells, the cells were placed on WPGN1K2 medium (selection medium) containing 20 mg / l geneticin as a drug. When Agrobacterium began to grow again from the petiole slice, it was transplanted to a WPGN1K2 medium supplemented with 500 mg / l Kraforan and sterilized. In addition, when the callus browning changes significantly due to the influence of the drug for sterilization and the drug for selection, the petiole slice was transplanted to the WPGN1K2 medium containing no drug to promote the growth of the callus. The appearance of transformed cells was evaluated using the reporter gene sGFP expression as an indicator.

  FIG. 4 shows the fluorescence of sGFP that appears as a result of introducing the sGFP gene into the petals of cultured aurene and cultured shoots. The fluorescence of sGFP was obtained by irradiating the callus formed on the petiole slice with excitation light (blue light), and observing the emitted green fluorescence (510 nm) with a stereoscopic fluorescence microscope (OLYMPUS SZX16).

  As shown in the figure, it was found that the sGFP gene was introduced into the callus formed on the petiole of the plant of the genus Oren, and a transformant was obtained from the petiole.

(Example 3: Transformation of cultured roots)
Transformation was performed by the following method using a cultured root of a plant belonging to the genus Auren (Coptis japonica Makino).

(vector)
Similar to Example 1, Agrobacterium tumefaciens LBA4404 is a binary vector pBI-sGFP having a kanamycin resistance gene (nptII) which is a marker gene for selecting transformed cells and a sGFP gene serving as a reporter for transformed cells on T-DNA. The fungus was transformed and used for the experiment.

(Callus induction)
A section about 5 mm long was prepared from the cultured root of aurene, planted in a WPGN1K2 solid medium supplemented with 2% sucrose, and cultured in the dark at 20 ° C. for 30 days.

(Agrobacterium infection)
Agrobacterium grown in an LB liquid medium at 28 ° C. in the dark was used as the bacterial solution. The cultured root slices that have been cultured and callus formed on both ends thereof are put into a WPGN1K2 liquid medium containing 2% sucrose, an appropriate amount of Agrobacterium solution is added, and 1 million to 1 per 1 ml of the medium is added. It was prepared to become 10 million Agrobacterium, and infection treatment was performed at 28 ° C. for 30 minutes while shaking at 60 rpm in the dark.

(Selection of sterilized and transformed callus)
After completion of the infection treatment, the cultured root slices infected with Agrobacterium were placed on a WPGN1K2 solid medium (sterilization medium) containing 500 mg / l Kraforan, and sterilization treatment was performed. Subsequently, in order to select the transformed cells, the cells were placed on WPGN1K2 medium (selection medium) containing 20 mg / l geneticin as a drug. When Agrobacterium began to grow again from the cultured root section, it was transplanted to a selective medium supplemented with 500 mg / l Kraforan and sterilized. Moreover, when the callus browning change was remarkable due to the influence of the drug for sterilization and the drug for selection, the cultured root slice was transplanted to a WPGN1K2 medium containing no drug to promote callus growth. The appearance of transformed cells was evaluated using the reporter gene sGFP expression as an indicator.

  FIG. 5 is a diagram showing the fluorescence of sGFP that appears as a result of introducing the sGFP gene into cultured roots of the plant belonging to the genus Oren. The fluorescence of sGFP was obtained by irradiating the callus formed on the cultured root slice with excitation light (blue light), and observing the emitted green fluorescence (510 nm) with a stereoscopic fluorescence microscope (OLYMPUS SZX16).

  As shown in the figure, it was found that the sGFP gene was introduced into the callus formed on the cultured root of the plant belonging to the genus Oren, and a transformant was obtained from the cultured root.

Next, Table 1 shows the transformation efficiency obtained as a result of Examples 1 to 3. The transformation efficiency was calculated by dividing the number of sections in which sGFP fluorescence was observed by the number of sections subjected to infection and multiplying by 100. The sGFP fluorescence was counted as 1 if observed in any part of the callus formed in one section. For sGFP fluorescence, the non-recombinant callus was observed before observing the test section, and the test section was observed after setting the excitation light and exposure conditions such that no fluorescence was observed in the non-recombinant callus.
In addition, as shown in FIGS. 2, 4, and 5, in fact, fluorescence is observed in several places, and the obtained recombinant cells are one or more per section.

  From the results shown in Table 1, transformation was performed at a transformation efficiency of 27 to 70% by infecting Agrobacterium with a callus subcultured in WPGN1K2 medium or a tissue culture-derived section cultured in WPGN1K2 medium. The body was shown to be obtained.

  As described above, a plant transformant of the genus Oren was able to be produced with high efficiency. In addition, since the method of the present invention uses a sterilized tissue culture, it is understood that the method is not affected by the time when the plant belonging to the genus Orenum can be collected, compared with the conventional method.

(Example 4: Production of transformant introduced with 6-OMT gene)
Using the plant belonging to the genus Auren (Coptis japonica Makino) as a material, transformation was performed by the following method.

(vector)
A cassette in which a norcoclaurin-6-O-methyltransferase (6-OMT) gene is linked downstream of a plant high expression promoter (EL2-35S), and a kanamycin resistance gene (marker gene for selecting transformed cells) A binary vector having NptII) or hygromycin resistance gene (hpt) on T-DNA was transformed into Agrobacterium tumefaciens LBA4404 and used in the experiment.

(Induction of callus that infects Agrobacterium)
The petioles of the sterilized oren field-cultivated plant were placed on a WPG medium (WPGHF medium) without addition of a plant growth regulator, and cultured in the dark at 20 ° C. for 9 days to induce callus. The composition of the WPG medium used for callus induction is as follows: 30 g / l sucrose, 10 mg / l L-glutamine, 1 mg / l NAA, 2 mg / l KIN, 2.5 g / l gellite, pH 5.7 .

(Agrobacterium infection)
A solution of Agrobacterium grown in YEB liquid medium at 25 ° C. in the dark was used. The petiole slice containing the callus is placed in a WPGN1K2 liquid medium, and an appropriate amount of Agrobacterium solution is added to prepare 1 to 10 million Agrobacterium per ml of the medium. The cells were co-cultured at 25 ° C. for 3 days with shaking.

(Selection of sterilized and transformed callus)
After completion of co-cultivation, the petiole sections infected with Agrobacterium were placed in sterilized water (Klaforan solution) containing 500 mg / l Kraforan, shaken at 100 rpm at 20 ° C. for 2 days in the dark, and then 500 mg / L It was placed on a WPGN1K2 solid medium (sterilization medium) containing 1 kuraforan and sterilized.

  Subsequently, in order to select transformed cells, the cells were placed on WPGN1K2 medium (selection medium) containing 150 mg / l kanamycin or 25 mg / l hygromycin as a drug. When Agrobacterium began to grow again from the section, it was transplanted to a WPGN1K2 medium (sterilization medium) supplemented with 500 mg / l Kraforan, and sterilized. Moreover, when the browning of the callus piece was remarkable due to the influence of the drug for sterilization and the drug for selection, the callus piece was transplanted to a WPGN1K2 medium containing no drug to promote callus growth. The confirmation of 6-OMT gene is performed by extracting DNA from callus or transformed plant body using DNeasy (QIAGEN) according to the conventional method, and specific for high expression promoter (EL2-35S) and 6-OMT gene. The PCR was performed using a primer set. Then, the sequence from the introduced EL2-35S to the 6-OMT gene was confirmed by electrophoresis. Similarly, the kanamycin resistance gene (nptII) and the hygromycin resistance gene (hpt) were confirmed by PCR using a marker gene specific primer set.

  FIGS. 6A and 6B are diagrams showing callus of a plant belonging to the genus Oren, into which a 6-OMT gene has been introduced. It was confirmed that the 6-OMT gene was introduced into the callus shown in FIG. Moreover, FIG. 6C is a figure which shows the 6-OMT gene introduction | transduction transformed plant body re-differentiated from the callus of FIG. 6B.

  As shown in FIGS. 6A to 6C, a genetically modified plant via callus was produced.

  7A and 7B are diagrams showing that the 6-OMT gene was introduced into the plant of the genus Oren. FIG. 7A is a diagram showing confirmation of introduction of a sequence from number EL2-35S to the 6-OMT gene, with the number 4 being the callus shown in FIG. 6A. FIG. 7B is a diagram showing confirmation of the introduction of the sequence from EL2-35S to the 6-OMT gene, with the number 2 being the callus shown in FIG. 6B.

  As shown in FIGS. 7A and 7B, plant transformants of the genus Oren were introduced into which the 6-OMT gene was introduced.

  As described above, a plant transformant of the plant belonging to the genus Oren that is transformed by introduction of the 6-OMT gene could be produced.

Claims (5)

A sterilization process for sterilizing tissues and organs of the genus Oren,
Plant tissue culture induction step of culturing the sterilized tissue / organ and inducing a plant tissue culture from the tissue / organ;
A subculturing step of subculturing the induced plant tissue culture;
A callus induction step of inducing callus from the induced or subcultured plant tissue culture;
Introducing a gene into a callus derived from the plant tissue culture, and transforming the callus, and
A method for producing a plant transformant characterized by the above. In the subculture step, the plant tissue culture is aseptically cultured,
In the transformation step, the callus is redifferentiated into a plant tissue or organ.
The method for producing a plant transformant according to claim 1. In the subculture process and the callus induction process, it is cultured in a solid medium containing auxin and cytokinin.
The production method of the plant transformant of Claim 1 or 2 characterized by the above-mentioned. In the transformation step, the gene is introduced by at least one of the Agrobacterium method, the polyethylene glycol method, the electroporation method, and the particle gun method.
The method for producing a plant transformant according to any one of claims 1 to 3.   A plant transformant of the genus Auren, which is transformed by introduction of a norcoclaurine-6-O-methyltransferase (6-OMT) gene.