JP2014180220A - Preparation method for transformed cell of sonchus plants - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently preparing a transformed cell of Sonchus plants.SOLUTION: The invention relates to a preparation method for a transformed cell of Sonchus plants which comprises: an infection process of infecting Agrobacterium bacteria containing a plasmid comprising a target gene or a fragment thereof to a callus obtained by culturing a tissue derived from Sonchus plant for two to seven weeks under callus inductive conditions; and a selective culture process of selectively growing the callus which obtains the target gene.

Description

The present invention relates to a method for producing a transformed cell of a Sonchus plant.

Natural rubber cultivates rubber-producing plants such as Hevea siliensis of Euphorbiaceae and Indian rubber tree of mulberry plant (Ficus elastica), biosynthesizes natural rubber with milk duct cells of the plant body, and the natural rubber It is obtained by manually collecting rubber from plants.

At present, the natural rubber used in industrial rubber products has para rubber tree as its sole source. Para rubber tree is a plant that can grow only in limited areas such as Southeast Asia and South America. Furthermore, it takes about seven years for para rubber tree to become an adult tree from which rubber can be collected from planting, and the period during which natural rubber can be collected is 20 to 30 years. In the future, demand for natural rubber is expected to increase mainly in developing countries. However, for the reasons described above, it is difficult to significantly increase the production of natural rubber using para rubber tree. For this reason, there is a concern about the depletion of natural rubber resources, and a stable supply source of natural rubber (isoprenoid) other than adult rubber trees is desired.

The Sonchus plant grows naturally in various parts of the world such as European countries and Asia such as Japan, and since it is a herbaceous plant, it grows quickly, and an emulsion containing natural rubber can be obtained from its stem and root. Therefore, the Sonchus genus plant has the potential to be a source of stable natural rubber (isoprenoid) other than adult trees of Para rubber tree.

On the other hand, with the recent development of genetic engineering, it has become possible to modify traits by introducing a preferred foreign gene into a natural plant body. Methods for introducing foreign genes into plants include the introduction of genes by infecting plant cells with Agrobacterium, which is one of the plant pathogens (Agrobacterium method), and gold particles carrying the genes as particles A method of shooting into a plant cell with cancer is known.

An example of successful production of a transformant of Para rubber tree, a rubber-producing plant, by the Agrobacterium method has been reported. However, as described above, the production area of para rubber tree is limited, and it takes about 7 years to collect rubber. Moreover, the applicability and application conditions of the Agrobacterium method vary depending on the plant, and the application conditions of the Agrobacterium method have only been studied in some plants such as rice.

When using a Sonchus plant as a source of new natural rubber, it is desirable to create a transformed cell of the Sonchus plant in order to increase the production of isoprenoids. There have been no reports of examples of gene introduction, and no attempt has been made to apply the Agrobacterium method to plants of the genus Sonchus.

An object of the present invention is to solve the above-mentioned problems and to provide a method for efficiently producing a transformed cell of a Sonchus genus plant.

As a result of intensive studies, the present inventors have succeeded in producing a transformed cell of a Sonchus genus plant. In order to increase the efficiency of gene transfer by examining the conditions for more efficiently creating transformed cells of the Sonchus plant, the infectivity of Agrobacterium and the state of the callus on the side receiving the gene Was found to be important. As a result of further studies based on this finding, various findings were found and the present invention was completed.
That is, the present invention infects callus obtained by culturing a tissue derived from a Sonchus plant under callus inducing conditions for 2 to 7 weeks with an Agrobacterium containing a target gene or a plasmid containing a fragment thereof. The present invention relates to a method for producing a transformed cell of a Sonchus genus plant, which has an infection step and a selective culturing step for selectively growing a callus that has acquired the target gene.

In the infection step, it is preferable to infect callus with Agrobacterium in an amino acid-containing medium.

The amino acid is preferably aspartic acid and / or glutamine.

In the infection step, it is preferable to infect callus with Agrobacterium in a medium that does not use ammonium nitrate as a nitrogen source in the medium.

In the infection step, it is preferable to infect callus with Agrobacterium in an acetosyringone-containing medium.

In the infection step, it is preferable to infect callus with Agrobacterium in a medium containing casamino acid.

In the above infection process, the number of Agrobacterium corresponding to 10 to 50 mL of an Agrobacterium suspension solution having an absorbance (OD 600) measured at 600 nm of 0.01 to 0.4 per 6 g of callus. It is preferable to contact bacteria belonging to the genus Bacteria.

In the above infection step, it is preferable that the coexistence time of Agrobacterium and callus is 0.5 to 60 minutes.

In the selective culture step, the culture temperature is preferably 20 to 28 ° C.

It is preferable that the Sonchus genus plant is Nochusi (Sonchus oleraceus).

The method for producing a transformed cell of a Sonchus plant of the present invention contains a plasmid containing a target gene or a fragment thereof in a callus obtained by culturing a tissue derived from a Sonchus plant under a callus-inducing condition for 2 to 7 weeks. Therefore, a transformed cell of a Sonchus genus plant can be produced efficiently because it has an infection step of infecting the genus Agrobacterium and a selective culture step of selectively growing the callus that has acquired the target gene.

The method for producing a transformed cell of the genus Sonchus of the present invention (hereinafter also referred to as the method of the present invention) comprises culturing a tissue (tissue fragment) derived from a genus Sonchus under callus-inducing conditions for 2 to 7 weeks. The obtained callus has an infection step of infecting a genus Agrobacterium containing a plasmid containing the target gene or a fragment thereof, and a selective culture step of selectively growing the callus that has acquired the target gene.

In the present invention, callus means an undifferentiated plant cell or an undifferentiated plant cell mass.

The plant of the genus Sonchus is not particularly limited, and examples thereof include the genus Sonchus such as Sonchus oleraceus, Sonchus asper, Beech horn (Sonchus brachytus), and Taiwan honeywort (Sonchus arvensis). Of these, Songe oleaceus is preferable.

Para rubber tree can grow in Southeast Asia and South America, but the genus Sonchus is a native species in Europe, Asia, and other parts of the world. Can be cultivated in Furthermore, Para rubber tree takes about 7 years from planting to collecting rubber, but Nogeshi is an herbaceous plant and is an annual grass, which is advantageous in terms of faster growth.

(Guidance process)
First, a callus preparation method (induction step) will be described.
In the induction step, for example, callus is induced by culturing a tissue piece (tissue) of a Sonchus genus plant (hereinafter also simply referred to as a plant) in an induction medium containing a plant growth hormone and a carbon source.

The tissue piece is not particularly limited, but is preferably at least one selected from the group consisting of leaves, stems, roots, buds, petals, cotyledons, hypocotyls, cocoons, and seeds. Of these, leaves and stems are preferred.

In the induction step, first, the surface of the plant tissue piece is washed. When using the tissue inside the plant as the tissue piece, for example, it may be washed with a sachet, but may be washed with water containing about 0.1% of a surfactant. When using leaves or the like, the surface is preferably washed with a soft sponge.

Next, the tissue piece is sterilized or sterilized. Sterilization or sterilization can be performed using a known disinfectant or sterilant, and ethanol, benzalkonium chloride, and an aqueous sodium hypochlorite solution are preferable.

Next, callus is induced by culturing the sterilized or sterilized tissue piece in an induction medium containing a plant growth hormone and a carbon source. The induction medium may be liquid or solid, but solid culture is preferable because it is easily placed on the medium and cultured for callus. In addition, when the induction medium is a liquid medium, stationary culture or shaking culture may be performed.

Examples of plant growth hormones include auxin plant hormones and / or cytokinin plant hormones.

Auxin plant hormones include 2,4-dichlorophenoxyacetic acid, naphthalene acetic acid, indolebutyric acid, indoleacetic acid, indolepropionic acid, chlorophenoxyacetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4,5-trichlorophenoxyacetic acid, parachloro Examples include phenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, 4-fluorophenoxyacetic acid, 2-methoxy-3,6-dichlorobenzoic acid, 2-phenyl acid, picloram, picolinic acid and the like. Of these, 2,4-dichlorophenoxyacetic acid, naphthalene acetic acid and indolebutyric acid are preferable, 2,4-dichlorophenoxyacetic acid and naphthalene acetic acid are more preferable, and naphthalene acetic acid is still more preferable.

Examples of cytokinin plant hormones include benzyladenine, kinetin, zeatin, benzylaminopurine, isopentinylaminopurine, thidiazuron, isopentenyladenine, zeatin liposide, dihydrozeatin and the like. Of these, benzyladenine, kinetin and zeatin are preferable, benzyladenine and kinetin are more preferable, and benzyladenine is more preferable.

The carbon source is not particularly limited, and examples thereof include saccharides such as sucrose, glucose, trehalose, fructose, lactose, galactose, xylose, allose, talose, gulose, altrose, mannose, idose, arabinose, apiose and maltose. Further, sugar alcohols such as erythritol, xylitol, mannitol, sorbitol, and lactitol may be used. Of these, sucrose and glucose are preferable, and sucrose is more preferable.

As an induction medium, White's medium (described in Plant Cell Engineering Introduction (Academic Publishing Center) p20-p36), Heller's medium (Heller R, Bot. Biol. Veg. Paris 14 1-223 (1953)), SH medium (Schenk and Hildebrandt's medium), MS medium (Murashige and Skoog's medium) (described in Plant Cell Engineering Introduction (Academic Publishing Center) p20-p36), LS Medium (Linmeier and Skoog Medium) (Introduction to Plant Cell Engineering (Academic Society) (Publishing center) p20 to p36), Gamborg medium, B5 medium (introduction to plant cell engineering (conference publication center) p20 to p36), MB medium, WP medium (Wooden Plant: for woody plants) and other basic media Or change the composition of the basic medium A medium obtained by adding a plant growth hormone to a base medium such as a modified basic medium may be used. Especially, what added plant growth hormone to MS culture medium, B5 culture medium, and WP culture medium is preferable. In addition, auxin-type plant hormones and cytokinin-type plant hormones are preferably included because they are suitable for maintaining callus and promoting cell division.

The induction medium may contain at least one selected from the group consisting of jasmonic acid and monoterpene compounds.
Examples of the monoterpene compound include D-limonene, α-pinene, β-pinene, l-menthol, geraniol, caran, pinane, myrcene, oschimen, and cosmene. Of these, D-limonene and α-pinene are preferable.

When the induction medium is a solid medium, a solidifying agent may be used to make the medium solid. The solidifying agent is not particularly limited, and examples thereof include agar, gellan gum, agarose, gellite, gelatin, silica gel and the like.

The composition of the suitable induction medium and the culture conditions vary depending on the plant species, and also depend on whether the medium is a liquid medium or a solid medium, but is usually the following composition (especially in the case of Nogeshi).

The concentration of the carbon source in the induction medium is preferably 0.1% by mass or more, more preferably 1% by mass or more. The concentration of the carbon source is preferably 10% by mass or less, more preferably 6% by mass or less, and still more preferably 3% by mass or less. In the present specification, the concentration of the carbon source means the concentration of saccharides.

The concentration of the auxin plant hormone in the induction medium is preferably 0 mg / l or more, more preferably 1 × 10 ⁻³ mg / l or more, still more preferably 0.05 mg / l or more, particularly preferably 0.5 mg / l. That's it. The concentration of the auxinic plant hormone is preferably 20 mg / l or less, more preferably 10 mg / l or less, and still more preferably 2.5 mg / l or less.

The concentration of the cytokinin plant hormone in the induction medium is preferably 0 mg / l or more, more preferably 1 × 10 ⁻³ mg / l or more, still more preferably 0.5 mg / l or more, particularly preferably 0.8 mg / l. That's it. The concentration of the cytokinin plant hormone is preferably 15 mg / l or less, more preferably 10 mg / l or less, still more preferably 3 mg / l or less, and particularly preferably 1.5 mg / l or less.

The pH of the induction medium is preferably 4.0 to 10.0, more preferably 5.6 to 6.5, and still more preferably 5.7 to 5.8. The culture temperature is preferably 0 to 40 ° C, more preferably 20 to 26 ° C. Cultivation may be performed in a dark place or in a bright place, but the illuminance is preferably 0 to 100000 lx, more preferably 0 to 0.1 lx. The culture time is 2 to 7 weeks (preferably 2 to 6 weeks, more preferably 3 to 5 weeks).
In the present specification, the pH of the solid medium means the pH of the medium to which all components except the solidifying agent are added. In the present specification, the dark place means that the illuminance is 0 to 0.1 lx, and the bright place means that the illuminance exceeds 0.1 lx.

In the case of a solid medium, the concentration of the solidifying agent in the induction medium is preferably 0.1% by mass or more, more preferably 0.2% by mass or more. The concentration of the solidifying agent is preferably 2% by mass or less, more preferably 1.1% by mass or less, and still more preferably 0.6% by mass or less.

Among the above-mentioned conditions, in particular, when the plant is Nogeshi, the auxin type plant hormone is naphthalene acetic acid, the concentration thereof is 0.5 to 2.5 mg / l, the cytokinin type plant hormone is benzyladenine, and the culture temperature. Is particularly preferably 20 to 26 ° C.

As described above, callus can be induced by culturing a sterilized or sterilized tissue piece in the induction medium.

In the method of the present invention, callus obtained by culturing tissue derived from a plant of the genus Sonchus under callus inducing conditions (the above induction medium) for 2 to 7 weeks is used. The culture period from the start of the culture of the Sonchus genus plant under the callus-inducing condition to the infection with Agrobacterium is not particularly limited as long as it is within a period of 2 to 7 weeks. It can be appropriately determined in consideration of the type of plant, the type and amount of plant tissue (tissue piece) used as a callus-inducing material, the amount of Agrobacterium used for infection, and the like. Preferably, callus obtained by culturing a tissue derived from a Sonchus plant under a callus-inducing condition for 2 to 6 weeks, more preferably, a tissue derived from a Sonchus plant under a callus-inducing condition for 3 to 5 weeks. The obtained callus, more preferably, a callus obtained by culturing tissue derived from a plant of the genus Sonchus under callus-inducing conditions for 4 weeks is used.

The callus obtained by culturing a tissue derived from a Sonchus plant under a callus-inducing condition for 2 weeks is a culture obtained by transferring a tissue derived from a Sonchus plant to a callus induction medium (the above induction medium) for 2 weeks. It means the obtained callus.

(Agrobacterium preparation process)
In the method of the present invention, callus is infected with an Agrobacterium genus containing a plasmid containing a target gene or a fragment thereof (hereinafter sometimes referred to as a target gene or the like). Then, next, the preparation method (Agrobacterium genus preparation process) of Agrobacterium is demonstrated.

The Agrobacterium used in the present invention is not particularly limited as long as it contains an Agrobacterium that can introduce the contained plasmid into plant cells. However, Agrobacterium tumefaciens is not particularly limited. ) Is preferable. This is because the infection efficiency is good and it is widely used in the Agrobacterium method.

An Agrobacterium genus containing a plasmid containing a target gene or the like may be prepared using any conventionally known technique. For example, a target gene recombination intermediate vector in which a target gene or the like is incorporated into a plasmid capable of homologous recombination with a T-DNA region of a Ti plasmid possessed by Agrobacterium is produced. It may be introduced into the fungus. In addition, a target gene binary vector in which a target gene or the like is incorporated into a binary vector widely used in the Agrobacterium method may be introduced into the genus Agrobacterium.

In the present invention, the target gene means a target gene to be introduced into a plant of the genus Sonchus. The target gene is not particularly limited as long as it can change the genetic traits of the Sonchus genus plant as a result of being introduced into the Sonchus genus plant. It may be a gene, a gene derived from an organism other than the Sonchus genus plant, or an artificially prepared gene. The artificially prepared gene may be, for example, a chimeric gene obtained by connecting two or more types of genes, or a mutant gene obtained by mutating a gene possessed by any organism. As a mutated gene, for example, a part of the base sequence of DNA constituting the gene may be deleted or may be replaced. Moreover, what inserted the partial base sequence in the middle of this base sequence may be used.

Further, the target gene may be a structural gene or a regulatory region. For example, it may be a structural gene containing a transcriptional or translational control region such as a promoter or terminator. In addition, the gene of the control region may be any gene as long as it can function in the Sonchus plant into which the gene is introduced, and may be a gene derived from the same species as the Sonchus plant into which the gene is introduced. Needless to say, it may be a gene derived from an organism. As such a heterologous promoter, for example, a promoter commonly used in the field of gene recombination such as CaMV35 promoter and NOS promoter can be used.

The target gene introduced into the plant of the genus Sonchus may be the full length of the gene or a fragment. For example, a fragment consisting only of a functional domain of a structural gene may be introduced.

Examples of target genes to be introduced into Sonchus plants include genes that are involved in latex biosynthetic mechanisms and polyisoprene chain extension reactions, and function in latex production and molecular weight, and proteins contained in latex. , Saccharides such as inositol and quebrachitol, genes involved in the biosynthesis of tocotrienol, which is a kind of vitamin E and is also effective as a natural anti-aging agent, and these proteins, It is preferably a gene or the like that produces a saccharide, a tocotrienol mutant. In addition, by including a regulatory region such as a promoter that functions in a tissue-specific manner in these genes, the protein encoded by the target gene can be expressed in a specific tissue of a plant body.

The target gene and the like are preferably incorporated into a vector together with a marker gene and optionally a reporter gene.
Examples of the marker gene include drug resistance genes such as kanamycin resistance gene (nptII), hygromycin resistance gene (hptI), and bleomycin resistance gene. Examples of the reporter gene for confirming the expression position in the plant include luciferase gene, GUS (β-glucuronidase) gene, GFP (green fluorescent protein), RFP (red fluorescent protein) and the like.

In the Agrobacterium preparation process, Agrobacterium containing a plasmid containing the target gene and the like prepared as described above is cultured by a conventional method (for example, at a culture temperature of 20 to 35 ° C., at YEB). By shaking and culturing in a medium for 10 to 30 hours, the amount necessary for infecting callus can be prepared.

(Infectious process)
In the infection step, callus obtained by culturing tissue derived from a Sonchus plant under callus-inducing conditions for 2 to 7 weeks is infected with an Agrobacterium containing a target gene or a plasmid containing a fragment thereof.

In the infection process, callus obtained by culturing tissue derived from a plant of the genus Sonchus under callus inducing conditions for 2 to 7 weeks (callus obtained by the induction process) contains an agro containing a plasmid containing the target gene or a fragment thereof. Infect bacteria belonging to the genus Bacteria (Agrobacterium genus obtained through the Agrobacterium preparation process).

The infection process can be performed by a technique generally used in the Agrobacterium method. For example, Agrobacterium can be infected by suspending in an infection medium and immersing callus in the suspension. Then, after the immersion, the suspension and the callus may be separated with a filter paper or the like. In addition, although it may stand still and may be shaken during immersion, since it is easy to infect callus with Agrobacterium, it is preferable to shake.

The concentration of the Agrobacterium suspension used for infection should be determined appropriately in consideration of the type and growth activity of Agrobacterium, the culture period after callus induction, the immersion time, etc. Can do. For example, with respect to 6 g of callus, the absorbance (OD 600) measured at 600 nm is 0.01 to 0.4 (preferably 0.05 to 0.2, more preferably 0.08 to 0.12). It is preferable to contact Agrobacterium having the number of cells corresponding to 10 to 50 mL (preferably 20 to 40 mL, more preferably 25 to 35 mL) of the Agrobacterium suspension solution. Thereby, the number of Agrobacterium which infects callus can be optimized, and a transformed cell can be produced efficiently.

The coexistence time of Agrobacterium and callus in the infection process, that is, the time for contacting Agrobacterium and callus is preferably 0.5 to 60 minutes, more preferably 1 to 40 minutes, and more preferably 25 to 35 minutes. Is more preferable. Thereby, the number of Agrobacterium which infects callus can be optimized, and a transformed cell can be produced efficiently. In addition, this coexistence time means immersion time, for example, when callus is immersed in an Agrobacterium suspension.

As an infectious medium for suspending Agrobacterium, plant growth hormone was added to the above-mentioned basic medium or a basic medium such as a modified basic medium in which the composition of the basic medium was changed as necessary. You can use something. Among these, MS medium, LS medium, B5 medium, and WP medium are preferable, and MS medium is more preferable. As the plant growth hormone and carbon source, those similar to the above-mentioned induction medium can be preferably used, but as the carbon source, it is more preferable to use sucrose and glucose in combination.

The composition of a suitable infection medium varies depending on the plant species, but is usually the following composition (especially in the case of Nogeshi).

The concentration of the carbon source in the infection medium is preferably 0.1% by mass or more, more preferably 1% by mass or more, still more preferably 2% by mass or more, and particularly preferably 3% by mass or more. The concentration of the carbon source is preferably 10% by mass or less, more preferably 6% by mass or less, and still more preferably 4% by mass or less.

When sucrose and glucose are used in combination as a carbon source, the concentration of sucrose in the infection medium is preferably 0.1 to 10% by mass, more preferably 0.5 to 5% by mass, and still more preferably 1 to 4% by mass. It is. On the other hand, the concentration of glucose in the infection medium is preferably 0.01 to 5% by mass, more preferably 0.1 to 3% by mass, and still more preferably 0.5 to 1.5% by mass.

It is preferable that the infectious medium does not use ammonium nitrate as a nitrogen source because the callus can be easily infected with Agrobacterium. And it is preferable to use the culture medium which used potassium nitrate instead of ammonium nitrate. The concentration of potassium nitrate in the infection medium is preferably 100 to 600 mg / l, more preferably 250 to 500 mg / l, still more preferably 300 to 450 mg / l.

The infection medium is preferably an amino acid-containing medium because the callus state can be improved. Although it does not specifically limit as an amino acid, For example, aspartic acid, glutamine, glutamic acid, asparagine etc. are mentioned. Of these, aspartic acid and glutamine are preferable, and aspartic acid and glutamine are preferably used in combination.

The concentration of the amino acid in the infection medium is preferably 500 mg / l or more, more preferably 700 mg / l or more, and still more preferably 1000 mg / l or more. The concentration of the amino acid is preferably 5000 mg / l or less, more preferably 2000 mg / l or less, and still more preferably 1300 mg / l or less.

When aspartic acid and glutamine are used in combination, the concentration of aspartic acid in the infection medium is preferably 100 to 700 mg / l, more preferably 200 to 500 mg / l, still more preferably 250 to 400 mg / l. On the other hand, the concentration of glutamine in the infection medium is preferably 100 to 1500 mg / l, more preferably 500 to 1200 mg / l, still more preferably 700 to 1100 mg / l.

It is preferable that the infection medium is an acetosyringone-containing medium because callus is easily infected with Agrobacterium. The concentration of acetosyringone in the infection medium is preferably 0.1 to 30 mg / l, more preferably 1 to 20 mg / l, still more preferably 5 to 15 mg / l.

It is preferable that the infection medium is a casamino acid-containing medium because the callus state and callus growth can be improved. The concentration of casamino acid in the infection medium is preferably 50 to 600 mg / l, more preferably 100 to 500 mg / l, still more preferably 200 to 400 mg / l.

The pH of the infection medium is not particularly limited, but is preferably 4.0 to 10.0, and more preferably 5.0 to 6.0. The temperature for infection (temperature of the infection medium) is preferably 0 to 40 ° C, more preferably 20 to 36 ° C, and still more preferably 22 to 24 ° C. The infection process may be performed in the dark or in the light.

As described above, in the infection process, for example, the Agrobacterium obtained in the Agrobacterium preparation step is suspended in the infection medium, and the callus obtained in the induction process is suspended in the suspension. By soaking, callus can be infected with Agrobacterium. Then, after the immersion, the suspension and the callus are separated with a filter paper or the like, and the separated callus is used in the next co-cultivation step.

(Co-culture process)
In the co-cultivation step, for example, callus (callus infected with Agrobacterium) obtained in the infection step is cultured in a co-culture medium. Thereby, a gene fragment such as a target gene introduced into callus by infection is incorporated into a gene of a plant cell, and a stable transformed cell can be obtained.

The co-cultivation medium may be liquid or solid, but solid culture is preferable because stable transformed cells can be obtained by placing the medium on the medium and culturing. In addition, when the co-culture medium is a liquid medium, stationary culture or shaking culture may be performed.

As the co-culture medium, a medium obtained by adding a plant growth hormone to the base medium such as the above-mentioned basic medium or a modified basic medium obtained by changing the composition of the basic medium as necessary may be used. Specifically, a medium similar to the above-described infection medium can be suitably used in the same preferred embodiment.

When the coculture medium is a solid medium, a solidifying agent may be used to make the medium solid as in the case of the induction medium.

The culture temperature is preferably 0 to 40 ° C, more preferably 10 to 36 ° C, still more preferably 20 to 28 ° C, and most preferably 22 to 24 ° C. The culture may be performed in a dark place or in a light place, but the culture is preferably performed in a dark place, and the illuminance in the dark place is preferably 0 to 0.1 lx. The culture time is not particularly limited, but it is preferable to culture for 2 to 4 days.

In the case of a solid medium, the concentration of the solidifying agent in the co-culture medium is preferably 0.1% by mass or more, more preferably 0.2% by mass or more. The concentration of the solidifying agent is preferably 2% by mass or less, more preferably 1.1% by mass or less, and still more preferably 0.6% by mass or less.

As described above, in the co-cultivation step, the callus obtained by the infection step (callus infected with Agrobacterium) is cultured in the co-culture medium, so that the target gene introduced into the callus by infection, etc. This gene fragment is incorporated into a plant cell gene, and a stable transformed cell can be obtained. The callus (mixture of transformed callus and non-transformed callus) obtained by this co-cultivation process is used in the next selective culture process.

(Selective culture process)
The selective culturing step can be performed by a technique generally used in the Agrobacterium method. By this step, transformed callus and untransformed callus can be selected.

In the selective culturing step, first, in order to sterilize Agrobacterium, a medium obtained by adding carbenicillin to a base medium such as the above basic medium or a modified basic medium obtained by changing the composition of the basic medium is used. Use to wash the callus (mixture of transformed and untransformed callus) obtained by the co-cultivation process. Prior to sterilization, callus (transformed callus) obtained in advance by a co-cultivation step with a base medium such as the basic medium described above or a modified basic medium obtained by changing the composition of the basic medium, The non-transformed callus mixture) may be washed.

The callus sterilized with carbenicillin is then cultured in a selective culture medium. The culture conditions in the selective culture step are not particularly limited as long as the transformed cells (callus that has acquired the target gene) can grow selectively.

The selective culture medium may be liquid or solid. In addition, when the selective culture medium is a liquid medium, static culture or shaking culture may be performed.

As a selective culture medium, a substance corresponding to a marker gene (a marker gene introduced together with a target gene) is added to a base medium such as the above basic medium or a modified basic medium obtained by changing the composition of the basic medium. What you add can be used. Especially, what added the substance corresponding to the said marker gene to MS culture medium, LS culture medium, B5 culture medium, and WP culture medium is preferable, and what added the substance corresponding to the said marker gene to MS culture medium is more preferable. Carbenicillin may be added as necessary. Moreover, you may add a plant growth hormone as needed. As the plant growth hormone and carbon source, those similar to the above induction medium can be preferably used.

Although it does not specifically limit as a substance corresponding to the said marker gene, Those skilled in the art can select suitably according to the marker gene used. For example, when a kanamycin resistance gene is used as the marker gene, callus (callus sterilized by carbenicillin (mixture of transformed callus and untransformed callus)) is added to the medium. Can be grown in the medium because the kanamycin resistance gene is introduced together with the target gene, but the non-transformed callus grows in the medium. There is nothing. In this way, by selectively cultivating a mixture of transformed callus and non-transformed callus in a medium to which a substance corresponding to the marker gene is added, the transformed callus is selectively grown. Can do.

When the selective culture medium is a solid medium, the medium may be made solid using a solidifying agent as in the case of the induction medium.

The pH of the selective culture medium is not particularly limited, but is preferably 5.0 to 7.0, and more preferably 5.6 to 6.5. The culture temperature is preferably 0 to 40 ° C, more preferably 10 to 36 ° C, still more preferably 20 to 28 ° C, and most preferably 22 to 24 ° C. The culture may be performed in a dark place or in a light place, but the culture is preferably performed in a dark place, and the illuminance in the dark place is preferably 0 to 0.1 lx. The culture time is not particularly limited, but subculture is preferably performed every 1 to 4 weeks.

In the case of a solid medium, the concentration of the solidifying agent in the selective culture medium is preferably 0.1% by mass or more, more preferably 0.2% by mass or more. The concentration of the solidifying agent is preferably 2% by mass or less, more preferably 1.1% by mass or less, and still more preferably 0.6% by mass or less.

As described above, in the selective culture process, the callus obtained by the co-cultivation process (a mixture of transformed callus and non-transformed callus) is washed with a carbenicillin-containing solution, and Agrobacterium is sterilized. To do. Next, the callus sterilized with carbenicillin is cultured in a selective culture medium, so that the transformed callus can be selectively grown. The transformed callus and the non-transformed callus are separated. Can be selected.

For example, in the selective culturing step, the gene transfer efficiency is obtained by culturing the callus obtained by the co-cultivation step (a mixture of transformed callus and non-transformed callus). It can be calculated by counting the number, that is, the number of transformed callus. Specifically, it can be calculated by the method described in the examples.

By the method of the present invention described above, a transformed cell (transformed callus) of a Sonchus plant can be efficiently produced.

(Creation of transformed plants)
Next, a method for producing a transformed plant will be described. For the preparation of the transformed plant, those skilled in the art can appropriately change the conditions using the transformed cells (transformed callus) prepared by the method of the present invention according to the description in the present specification and the technical common sense at the time of filing. Can be done.

Briefly, the plant body may be regenerated by inducing somatic embryos and shoots from the transformed cells prepared by the method of the present invention, elongating and rooting the shoots as follows.

(Regeneration induction process)
In the regeneration induction step, somatic embryos and shoots are formed by culturing callus (transformed callus) in a regeneration induction medium containing a plant growth hormone and a carbon source. Since shoots can be stably formed by inducing (forming) somatic embryos from callus and culturing somatic embryos, the culture conditions of the regeneration induction process should be conditions that can induce somatic embryos from callus. There is no particular limitation.

As the regeneration induction medium, a medium obtained by adding plant growth hormone to a base medium such as the above-mentioned basic medium or a modified basic medium obtained by changing the composition of the basic medium may be used. Especially, what added plant growth hormone to MS culture medium, LS culture medium, B5 culture medium, and WP culture medium is preferable, and what added plant growth hormone to MS culture medium is more preferable. As the plant growth hormone and carbon source, those similar to the above induction medium can be preferably used. In addition, auxin-type plant hormones and cytokinin-type plant hormones are preferably included because they are suitable for induction of somatic embryos.

The pH of the regeneration induction medium is not particularly limited, but is preferably 4.0 to 10.0, and more preferably 5.6 to 6.5. The culture temperature is preferably 0 to 40 ° C, more preferably 20 to 36 ° C. The culture may be performed in the dark or in the light. However, the culture is preferably performed in the light for 10 to 16 hours out of 24 hours. Is more preferable. The culture time is not particularly limited, but is preferably cultured for 1 to 10 weeks, more preferably 5 to 10 weeks.

As described above, in the regeneration induction step, somatic embryos and shoots can be formed by culturing callus (transformed callus) in the regeneration induction medium. The chute formed by this regeneration induction process is used for the next extension process. The timing for moving to the next stretching step is preferably after germination is visually recognized from the shoot and after confirming that it has grown stably.

(Extension process)
In the elongation step, the shoots are elongated by culturing the formed shoots in an elongation medium.

In the extension step, for example, the shoots formed by the regeneration induction step are cultured in an extension medium to extend the shoots. The extension medium may be liquid or solid, but solid culture is preferable because it is easy to extend the shoot by placing it on the medium and culturing. Further, when the extension medium is a liquid medium, static culture may be performed, or shaking culture may be performed.

As the extension medium, the above-mentioned basic medium or a modified basic medium in which the composition of the basic medium is changed may be used. However, for the reason that the shoot can be suitably extended, the extension medium contains the plant growth hormone. It is preferably a medium that does not contain, and more preferably an MS medium that does not contain plant growth hormone. In addition, as a carbon source, the thing similar to the said induction medium can be used conveniently.

The pH of the extension medium is not particularly limited, but is preferably 4.0 to 10.0, and more preferably 5.6 to 6.5. The culture temperature is preferably 0 to 40 ° C, more preferably 20 to 36 ° C. The culture may be performed in the dark or in the light. However, the culture is preferably performed in the light for 10 to 16 hours out of 24 hours. Is more preferable. The culture time is not particularly limited, but is preferably cultured for 1 to 10 weeks, more preferably 5 to 10 weeks.

As described above, in the extension step, the shoots can be extended by culturing the formed shoots in the extension medium. Further, in this extension step, not only the shoot extends but also a new shoot is formed. The chute extended by this extension process is used for the next rooting process. The timing for moving to the next rooting step is preferably after the chute is stretched to a size of about 2 to 3 cm.

(Rooting process)
In the rooting step, the elongated shoots are rooted by culturing in a rooting medium.

In the rooting step, for example, the shoots elongated in the elongation step are cultured in a rooting medium for rooting. The rooting medium may be liquid or solid, but solid culture is preferable because it is easy to root by placing it on the medium and culturing. In addition, when the rooting medium is a liquid medium, static culture may be performed or shaking culture may be performed.

As the rooting medium, the above-mentioned basic medium or a modified basic medium obtained by changing the composition of the basic medium may be used. For the reason that rooting can be preferably performed, the rooting medium is a plant growth hormone. It is preferable that the medium does not contain B5 medium that does not contain plant growth hormone. In addition, as a carbon source, the thing similar to the said induction medium can be used conveniently. In addition, the composition of the extension medium and the rooting medium may be the same. In addition, when rooting has already been performed in the extension step, this rooting step may be omitted.

The pH of the rooting medium is not particularly limited, but is preferably 4.0 to 10.0, and more preferably 5.6 to 6.5. The culture temperature is preferably 0 to 40 ° C, more preferably 10 to 36 ° C, and still more preferably 10 to 25 ° C. The culture may be performed in the dark or in the light. However, the culture is preferably performed in the light for 10 to 16 hours in 24 hours, and the illuminance in the light is preferably 2000 to 25000 lx. The culture time is not particularly limited, but it is preferable to culture for 5 to 10 weeks.

As described above, in the rooting step, rooted shoots (young plants (transformed plants)) can be obtained by cultivating the elongated shoots in the rooting medium. It is done. The seedlings may be transplanted directly into the soil, but it is preferable that the seedlings are transferred to artificial soil such as verculite and acclimated before being transplanted into the soil.

As described above, in the present invention, a transformed cell (transformed callus) of a Sonchus plant can be efficiently produced. In addition, by inducing somatic embryos from callus (transformed callus) and culturing somatic embryos, shoots can be stably formed, and the formed shoots can be elongated and rooted. The transformed callus can be stably regenerated into a plant (transformed plant).

The present invention will be specifically described based on examples, but the present invention is not limited to these examples.

Hereinafter, various chemicals used in the examples will be described together.
NAA: naphthalene acetic acid BA: benzyladenine acetosyringone: 4-hydroxy-3 ′, 5′-dimethoxyacetophenone casamino acid manufactured by Tokyo Chemical Industry Co., Ltd .: Bacto ^™ Casano Acids manufactured by Japan BD
Nogeshi: A plant that has been germinated aseptically from seeds of Nogeshi native to Kashiwa-ku, Kobe, Japan.

(Callus induction (induction process))
Leaves and stems were collected from nogeshi. Next, the surfaces of the collected leaves and stems were washed with running water, further washed with 70% ethanol, sterilized with a sodium hypochlorite solution diluted to about 5 to 10%, and washed again with running water.

Next, the sterilized leaf and stem tissues were inserted into an induction medium (solid medium) and cultured (induction process). The induction medium is MS medium (described in Plant Cell Engineering Introduction (Academic Publishing Center) p20-p36), naphthalene acetic acid (NAA), benzyladenine (BA), and sucrose at 1.0 mg / L and 0.1 mg / L, respectively. 3% by mass, adjusting the pH of the medium to 5.7-5.8, then adding gellan gum to 0.2% by mass, and autoclaving (121 ° C., 20 minutes) It was prepared by sterilization and cooling in a clean bench.

Nogeshi tissue pieces were inserted into an induction medium (solid medium) and cultured at 23 ° C. in the dark (0-0.1 lx) for 2 to 6 weeks to induce callus (undifferentiated cells) from the nogeshi tissue pieces. .

(Agrobacterium culture (Agrobacterium preparation process))
Agrobacterium (Agrobacterium tumefaciens) into which a binary vector into which a hygromycin resistance gene (hptI) that is a marker gene was inserted together with the target gene was cultured with shaking in a YEB medium at a culture temperature of 28 ° C. for 24 hours. Culturing until the absorbance measured at 600 nm (OD600) = about 1.0, collecting the cells by centrifugation, and diluting with a suspension solution (infectious medium described in Table 1) so that OD600 = 0.1 did. The absorbance was measured with a Nano Drop 2000 c manufactured by Thermo Scientific.

(Infection of Agrobacterium nogesicalus and co-cultivation (infection process, co-cultivation process))
First, the callus obtained by (callus induction (induction step)) was weighed. Next, 30 ml of the prepared Agrobacterium suspension (absorbance measured at 600 nm: 0.1) and 6 g of callus were placed in a 50 mL tube and shaken at 23 ° C. for 30 minutes (infection process). After shaking, the callus was collected, drained well, transplanted to a co-culture medium (the same medium (solid medium) as the infection medium described in Table 1), and placed in the dark (0-0) at 23 ° C. for 3 days. .1 lx). In Table 1, 2 weeks after callus induction means that calluses obtained by inserting nogeshi tissue pieces into an induction medium (solid medium) and culturing for 2 weeks under the above culture conditions were used. Further, “MS medium—NH ₄ NO ₃ ” means a medium obtained by removing NH ₄ NO ₃ from the MS medium. In Examples and Comparative Examples, KNO ₃ was added to the MS medium instead of removing NH ₄ NO ₃ from the MS medium.

(Selective culture of transformed callus (selective culture process))
The callus after co-culture was collected, washed 4 times with 30 mL of MS medium, and then washed with 30 mL of MS medium containing carbenicillin (500 mg / mL). After washing, the water was thoroughly drained and transplanted to MS medium (antibiotic concentration 10 mg / mL, solid medium, pH: 5.8) containing antibiotics (hygromycin) at 23 ° C. in the dark (0-0 .1 lx).
The evaluation of target gene introduction efficiency was performed as follows.
Every 2-3 weeks, subculture was carried out for 2 generations in the above-mentioned antibiotic-containing MS medium, the number of viable callus was counted, and the gene transfer efficiency was calculated.
Gene transfer efficiency (%) = number of viable callus (number) / number of initial cultures (number) × 100
Initial culture number = 60

(Adventitious embryo and shoot formation (regeneration induction process))
Next, somatic embryos and shoots were formed from the live callus (transformed callus) (regeneration induction step).
Specifically, auxin-type plant hormone NAA, cytokinin-type plant hormone BA, and saccharide sucrose are added to the MS medium to 0.01 mg / mL, 1 mg / mL, and 3% by mass, respectively. Medium was used. The pH was adjusted to 5.7. In addition, the gellan gum which is a solidification agent was added to the culture medium so that it might become 0.2 mass%. Then, using the prepared solid medium (sterilized), surviving calli (transformed callus) were cultured for 8 weeks under a culture temperature of 23 ° C. under illumination for 12 hours (10000 lx) for 24 hours. As a result, shoot formation was also observed after somatic embryo formation.

(Chute elongation (extension process))
The formed shoots were then transplanted into MS medium without plant growth hormone for shoot elongation. The pH of the medium was adjusted to 5.7. Using a solid medium (sterilized) containing 0.4% gellan gum, the cells were cultured for 8 weeks under a culture temperature of 23 ° C. and under illumination for 12 hours for 24 hours (10000 lx). Good shoot elongation was observed by culturing in a medium not containing plant growth hormone.

(Rooting (rooting process))
Next, for rooting, shoots grown to about 3 cm were transplanted into B5 medium containing no plant growth hormone. The pH of the medium was adjusted to 5.7. Using a solid medium (sterilized) containing 0.4% gellan gum, the cells were cultured for 8 weeks under a culture temperature of 16 ° C. under illumination for 12 hours for 24 hours (10000 lx). By culturing in a medium containing no plant growth hormone, rooting was observed well, and a transformed plant was obtained.

From the results of Table 1, callus obtained by culturing a tissue derived from a Sonchus plant under callus-inducing conditions for 2 to 7 weeks is infected with an Agrobacterium containing a target gene or a plasmid containing a fragment thereof. It has been found that a transformed cell of a Sonchus genus plant can be efficiently produced by a method having an infection process and a selective culture process for selectively growing a callus that has acquired the target gene.

Claims (10)

Infecting step of infecting callus obtained by culturing a tissue derived from a Sonchus plant under callus inducing conditions for 2 to 7 weeks with an Agrobacterium containing a plasmid containing the target gene or a fragment thereof, and the target A method for producing a transformed cell of a Sonchus genus plant, comprising a selective culturing step for selectively growing a callus having acquired a gene. The method for producing transformed cells of a Sonchus genus plant according to claim 1, wherein, in the infection step, callus is infected with Agrobacterium in an amino acid-containing medium. The method for producing a transformed cell of a Sonchus genus plant according to claim 2, wherein the amino acid is aspartic acid and / or glutamine. The transformation of a Sonchus genus plant according to any one of claims 1 to 3, wherein in the infection step, callus is infected with Agrobacterium in a medium that does not use ammonium nitrate as a nitrogen source in the medium. How to make a cell. The method for producing a transformed cell of a Sonchus genus plant according to any one of claims 1 to 4, wherein in the infection step, Agrobacterium is infected with callus in a medium containing acetosyringone. The method for producing a transformed cell of a Sonchus genus plant according to any one of claims 1 to 5, wherein in the infection step, a callus is infected with Agrobacterium in a casamino acid-containing medium. In the infection step, an agrobacterium having a number of cells corresponding to 10 to 50 mL of an Agrobacterium suspension solution having an absorbance (OD600) measured at 600 nm of 0.01 to 0.4 per 6 g of callus. A method for producing a transformed cell of a Sonchus genus plant according to any one of claims 1 to 6, wherein a bacterial bacterium is contacted. The method for producing a transformed cell of a Sonchus genus plant according to any one of claims 1 to 7, wherein the coexistence time of Agrobacterium and callus is 0.5 to 60 minutes in the infection step. . The method for producing a transformed cell of a Sonchus genus plant according to any one of claims 1 to 8, wherein, in the selective culturing step, a culture temperature is 20 to 28 ° C. The method for producing a transformed cell of a Sonchus genus plant according to any one of Claims 1 to 9, wherein the Sonchus genus plant is Nochusi (Sonchus oleraceus).