JP2010161989A - Method for preparing transformed cell of latex-producing plant, transformed plant, and method for preparing transformed plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stably and highly-efficiently preparing a transformed cell of a latex-producing plant by improving the efficiency of gene transduction into a callus derived from a latex-producing plant such as Hevea brasiliensis. <P>SOLUTION: There is disclosed a method for preparing a transformed cell of a latex-producing plant, wherein the method has an infection process of infecting a callus obtained by 5 to 9 weeks cultivation after callus induction of a tissue derived from a latex-producing plant with genus Agrobacterium bacteria containing a plasmid including a target gene or fragments thereof, wherein the genus Agrobacterium bacteria are characteristically in a logarithmic growth phase of bacteria. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for efficiently producing a transformed cell of a latex-producing plant, and a method for producing a transformed plant from the transformed cell produced by the method.

  Natural rubber is a polymer having elasticity, and is used widely and in large quantities in various applications as a main raw material for rubber products. Natural rubber is produced by collecting a latex secreted by a latex-producing plant such as rubber tree and subjecting it to desired processing. For this reason, rubber trees for recovering latex, particularly para rubber (Hevea brasiliensis), are planted commercially in tropical countries such as Thailand, Malaysia and Indonesia.

  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. In the field of natural rubber production, a plant having a desired trait such as a plant that can genetically improve a latex-producing plant and produce a higher-quality latex or a plant that can produce a larger amount of latex. The method of creating the body has been studied. Among them, molecular breeding by genetic recombination is a preferable technique, but in order to obtain a genetically modified plant body, it is necessary to go through a process of redifferentiation and gene introduction.

  Plant gene introduction methods include a method of introducing a gene by infecting a plant cell with a bacterium belonging to the genus Agrobacterium (Agrobacterium), which is one type of plant pathogen (Agrobacterium method), and a gold particle carrying a gene. A method of shooting into a plant cell with a particle gun (particle gun method) is mainly used (for example, see Patent Document 1).

  There are several examples of successful production of Para rubber tree transformants by the Agrobacterium method. For example, a method for producing a transformed para rubber tree by introducing a desired gene into a callus derived from an Agrobacterium genus using a vector system of a genus Agrobacterium and regenerating the plant from the plant tissue is disclosed. (For example, refer to Patent Document 2). In addition, there is a report that a transformant was obtained from callus derived from para rubber tree by the Agrobacterium method (for example, see Non-Patent Document 1).

JP 2005-130815 A

Japanese Patent No. 3289021

Blanc, 4 others, Plant cell Report, 2006, volume 24, pages 724-733.

  However, latex-producing plants such as Para rubber tree have problems that gene introduction is difficult and gene introduction efficiency is very poor compared to other types of plants. Even in the above-mentioned case, although efficiency improvement is being studied by adjusting the concentration of Agrobacterium when infecting callus, sufficient efficiency of gene introduction has not been achieved.

  An object of the present invention is to provide a method for improving the efficiency of gene transfer into a callus derived from a latex-producing plant such as para rubber tree, and stably and efficiently producing a transformed cell of a latex-producing plant.

  As a result of diligent research to solve the above problems, the present inventors have found that infectivity of Agrobacterium and the state of the callus on the side receiving the gene are important in order to increase gene transfer efficiency, We found that the balance between the two was particularly important. Furthermore, by using Agrobacterium in the logarithmic growth phase and infecting callus obtained by culturing for 5 to 9 weeks after callus induction, a higher gene transfer efficiency than ever has been achieved. The present inventors have found that transformed cells can be stably and efficiently produced and completed the present invention.

That is, the present invention
(1) A method for preparing a transformed cell of a latex-producing plant, wherein a plasmid containing a target gene or a fragment thereof is added to a callus obtained by culturing a tissue derived from a latex-producing plant after 5 to 9 weeks of callus induction. A method for producing a transformed cell of a latex-producing plant, comprising an infection step of infecting a bacterium belonging to the genus Agrobacterium, wherein the genus Agrobacterium is a logarithmically growing bacterium ,
(2) Furthermore, it has a growth step of growing callus infected with Agrobacterium in the infection step in a cell culture vessel, and the humidity in the cell culture vessel is adjusted to 50 to 70%. A method for producing a transformed cell of a latex-producing plant according to the above (1),
(3) The cell culture container sealed with a sealing member having bacteria impermeability and moisture permeability is installed in a constant temperature culture apparatus whose humidity is adjusted to 50 to 70%. (2) A method for producing transformed cells of the latex-producing plant according to the above,
(4) Before the callus obtained by culturing for 5 to 9 weeks after the callus induction used in the infection step is infected with Agrobacterium, stress resistance-inducing hormone may be added to all or part of the culture period. The method for producing a transformed cell of a latex-producing plant according to any one of (1) to (3), wherein the method is cultured in a containing medium,
(5) The callus infected with Agrobacterium is grown in a stress-resistance-inducing hormone-containing medium in the growth step, according to any one of (2) to (4), A method for producing transformed cells of latex-producing plants,
(6) The method for producing a transformed cell of a latex-producing plant according to (4) or (5), wherein the stress resistance-inducing hormone is abscisic acid,
(7) The method for producing a transformed cell of a latex-producing plant according to any one of (1) to (6), wherein the latex-producing plant is Para rubber tree (Hevea brasiliensis),
(8) A somatic somatic embryo is induced from a transformed cell obtained by the method for producing a transformed cell of a latex-producing plant according to any one of (1) to (7), and from the somatic somatic embryo A method for producing a transformed plant, characterized by regenerating a plant body,
(9) A transformed plant of Hevea brasiliensis produced by the method for producing a transformed plant according to (8),
The purpose is to provide.

  Since the method for producing a transformed cell of a latex-producing plant of the present invention can improve the gene transfer efficiency when the Agrobacterium method is used, the transformed cell of a latex-producing plant can be efficiently and stably produced. Can be created.

Reference Example 1, when culture of each number initial bacteria different Agrobacterium is a diagram showing the measured absorbance (OD ₆₆₀₎ at each culture time.

<Method for producing transformed cells of latex-producing plant>
The method for producing a transformed cell of a latex-producing plant of the present invention (hereinafter sometimes abbreviated as a method of producing a transformed cell) is a method for producing a transformed cell of a latex-producing plant, which is derived from a latex-producing plant. An infection step of infecting the callus obtained by culturing the tissue of 5 to 9 weeks after the callus induction with an Agrobacterium containing a plasmid containing the target gene or a fragment thereof, Is a logarithmically growing bacterium. By optimizing both the infectivity of Agrobacterium on the gene delivery side and the state of the callus on the gene receiving side, and balancing the two appropriately, Agrobacterium can be used very efficiently. The target gene possessed can be introduced into the callus.

  In the present invention, the latex-producing plant is not particularly limited as long as it is a plant in which latex (mainly polyisoprene) is contained in the milk ducts or cell gaps. For example, a para rubber tree, a rubber tree of Euphorbiaceae (Manihot glaziovii), Indian rubber tree of the mulberry family (Ficus elastica), Pana rubber tree (Castilloa elastica), Lagos rubber tree (Ficus lutea var.) Stag beetle (Dyera costulata), Zanzibar kurumii (Landolphia kirkii), huntsmia elastica (F ntumia elastica), Urceola elastica), Asteraceae guayule rubber (Parthenium argentatum), rubber dandelion (Taraxacum koko-saghyz), Gataperchamata palatamu (palatum) zapota), Cyptostegia grandiflora, Eucommia ulmoides, and the like. Among these, para rubber tree, sea rubber tree, Indian rubber tree and the like are preferable, and para rubber tree which is widely used as an industrial natural rubber raw material is more preferable.

[Preparation of callus]
In the method for producing a transformed cell of the present invention, callus obtained by culturing a tissue derived from a latex-producing plant for 5 to 9 weeks after callus induction is used. The culture period from callus induction to Agrobacterium infection is not particularly limited as long as it is within a period of 5 to 9 weeks. The types of latex-producing plants, the types of plant tissues used as materials for callus induction, The amount can be appropriately determined in consideration of the amount, the amount of Agrobacterium used for infection, and the like. Preferably, callus obtained by culturing for 6 to 9 weeks after callus induction is used.

  Callus induction on plant tissue for preparing a callus having redifferentiation ability may be performed by any conventionally known technique. For example, callus is induced from plant tissue by culturing a tissue derived from a latex-producing plant cut to an appropriate size in a callus induction medium (dedifferentiation induction medium) containing a hormone capable of promoting dedifferentiation. be able to. Specific examples of hormones that can promote dedifferentiation include cytokinin plant hormones and auxin plant hormones.

  The plant tissue that induces callus is not particularly limited as long as it is a tissue derived from a latex-producing plant, and various parts such as roots, stems, leaves, or flowers can be cut into appropriate sizes and used. . The plant tissue used in the present invention is preferably a bud, an immature fruit or a stem, and more preferably a cocoon taken from the bud, an immature seed taken from the immature fruit, or a formation layer taken from the stem.

Specifically, callus can be induced as follows.
First, the surface of the tissue of the latex producing plant is washed. When using the internal tissue of the plant as the plant tissue, for example, it may be washed with a scouring powder, or with water containing about 0.1% of a surfactant. When using leaves or the like, the surface may be washed with a soft sponge.
Next, the washed plant tissue is sterilized or sterilized. Sterilization or sterilization can be performed using a well-known disinfectant / sterilant, but it is preferable to use ethanol, benzaconium hydrochloride, sodium hypochlorite aqueous solution, or the like.

  After the treatment such as sterilization, the plant tissue is transferred to a callus induction medium and cultured. The callus induction medium can be prepared, for example, by adding a cytokinin plant hormone and / or an auxin plant hormone at a low concentration to a hormone-free plant cell culture medium generally used for plant cell culture. As the plant cell culture medium, White medium, Heller medium, SH medium (Schenk and Hildebrandt medium), MS medium (Murashige and Skoog medium), LS medium (Linsmaier and Skoog medium), Gamborg, B5, MB medium, WPM medium (LLOYD AND McCOWN'S Woody Plant Medium) and the like. The callus induction medium used in the present invention includes MS medium, MB medium, MS modified medium (medium in which the composition of the MS medium is changed), MB modified medium, or WPM medium, cytokinin plant hormone and / or auxin. A medium supplemented with a plant hormone is preferred.

As the cytokinin plant hormone, BAP (benzylaminopurine), 2iP (isopentinylaminopurine), kinetin (6-furfurylaminopurine), and zeatin can be used. Preferably, BAP (benzylaminopurine) is used. Purine) or KIN (Kinetin) can be used at 1 to 3 mg / L.
As auxinic plant hormones, pCPA (chlorophenoxyacetic acid), 2,4-D (dichlorophenoxyacetic acid), IAA (indoleacetic acid), IBA (indolebutyric acid), NAA (naphthaleneacetic acid), NOA (naphthoxyacetic acid) are used. be able to. Preferably, a combination of 0.3 to 1.0 mg / L of 2,4-D (dichlorophenoxyacetic acid) and about 1 mg / L of IAA (indoleacetic acid) or about 1 mg / L of NAA (naphthaleneacetic acid) Can be used.
More preferably, a cytokinin plant hormone and an auxin plant hormone can be used in combination. For example, BAP or KIN, 2,4-D, and IAA or NAA may be used in combination. it can.

In addition to cytokinin plant hormones and auxin plant hormones, substances that can promote callus induction may be added to the callus induction medium as appropriate. For example, sucrose may be added at a high concentration of 4 to 7 wt%, or coconut water may be added.
Further, as the callus induction medium, a liquid medium may be used, or a medium using gellite, agar or the like as a support for callus may be used.

  Callus can be induced by culturing plant tissue in a callus induction medium. Preferably, the pH of the callus induction medium is adjusted to 5.6 to 5.8, and the culture temperature is 26 to 28 ° C., and callus is induced in the dark.

  In the present invention, “after callus induction” means “after the plant tissue is cultured in the callus induction medium”. That is, in the method for producing a transformed cell of the present invention, callus obtained by transferring a tissue derived from a latex-producing plant to a callus induction medium and culturing for 5 to 9 weeks is infected with Agrobacterium. The reason why the gene transfer efficiency is improved by using the callus for 5 to 9 weeks after the callus induction is not clear, but the callus for 5 to 9 weeks after the callus induction is proliferating. In addition to the improvement in the infection efficiency of the genus Bacteria, the infected cells also proliferate well, and as a result, it is presumed that the gene transfer efficiency is significantly improved.

[Preparation of Agrobacterium]
In the method for producing a transformed cell 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).
The Agrobacterium used in the present invention is not particularly limited as long as it is capable of introducing 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.

  By culturing and proliferating Agrobacterium containing a plasmid containing the target gene and the like prepared in this manner by a conventional method, an amount necessary for infecting callus can be prepared.

  In the present invention, the target gene means a target gene to be introduced into a latex-producing plant. The target gene is not particularly limited as long as it can change the genetic traits of the latex-producing plant as a result of being introduced into the latex-producing plant. It may be a gene, a gene derived from an organism other than the latex-producing 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. The gene of the control region may be any gene as long as it can function in the latex-producing plant into which the gene is introduced, and may be a gene derived from the same species as the latex-producing 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 latex-producing plant 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 latex-producing plants include genes that are involved in latex biosynthesis mechanism and polyisoprene chain extension reaction, 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 selection markers include drug resistance genes such as kanamycin resistance gene (nptII), hyegromycin resistance gene (hptI), and bleomycin resistance gene. Examples of the reporter gene for confirming the expression position in the plant body include a luciferase gene and a GUS (β-glucuronidase) gene.

  In the method for producing transformed cells of the present invention, callus is infected with Agrobacterium in the logarithmic growth phase. Callus can be efficiently infected by using an Agrobacterium in the exponential growth phase that grows exponentially. This is presumably because the activity of the fungus is activated and the infectivity is increased by adjusting the time of the cells of the genus Agrobacterium to the logarithmic growth phase. In the present invention, it is preferable to use an Agrobacterium belonging to the callus infectivity in the middle of the logarithmic growth phase, which has a particularly high growth activity.

[Infection process]
Infection of callus obtained by culturing for 5 to 9 weeks after callus induction with a bacterium belonging to the genus Agrobacterium containing a plasmid containing a target gene or the like is performed by a method commonly used in the Agrobacterium method. be able to. For example, infection can be achieved by immersing callus in an Agrobacterium suspension. 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, callus can be infected for 1 second to 10 minutes in an Agrobacterium suspension prepared to 1 × 10 ⁷ to 1 × 10 ⁹ cells / mL.

  After soaking, callus is planted in a dedifferentiation-inducing hormone-free plant cell culture medium and cultured for 1 to 7 days, so that a gene fragment such as a target gene introduced into the callus by infection is incorporated into the gene of the plant cell. Stable transformed cells can be obtained. The culture during this time may be performed at about 25 to 28 ° C., similar to the callus culture, but may be performed at a temperature lower by about 2 to 8 ° C. than the callus culture temperature.

[Proliferation process]
By further growing the transformed cells obtained in this way, the desired transformed cells can be obtained more stably and at a high rate. In this growth step, in order to efficiently grow the transformed cells, it is carried out with a suitable plant cell culture medium at a general cell culture temperature of about 25 to 28 ° C. In general, the cells are cultured for about 2 to 5 months while being transplanted every 1 to 3 weeks.

  When a marker gene such as a drug resistance gene is introduced together with the target gene or the like, only transformed cells can be selected by growing in a plant cell culture medium to which a drug or the like is added in the growth step.

  In the growth step, in general, in order to prevent contamination due to contamination with germs or the like, the cell culture container is cultured with the body and the lid sealed with parafilm or the like. In the present invention, higher gene transfer efficiency can be achieved by culturing under humidity control conditions in which the humidity in the cell culture container is adjusted to an appropriate range. The humidity in the cell culture container is preferably adjusted to 50 to 70%, more preferably about 60%.

  Specifically, the humidity is adjusted to 50 to 70% in the cell culture container in which the main body and the lid of the cell culture container are sealed with a sealing member that is impermeable to bacteria and has moisture permeability. The humidity in the cell culture vessel can be adjusted to 50 to 70% by installing in the constant temperature culture apparatus. As such a sealing member, for example, a porous sealing member having an average pore diameter of 0.1 μm or less, preferably 0.08 μm or less is preferable. This is because, if the average pore size is 0.1 μm or less, water molecules, oxygen molecules, carbon dioxide molecules, etc. can permeate sufficiently while preventing permeation of bacteria and the like. As such a sealing member, for example, a highly permeable surgical tape (medical auxiliary tape) or the like can be used (see, for example, JP-A-2007-75176).

[Addition of stress resistance-inducing hormone]
In the present invention, the efficiency of gene transfer can be further increased by culturing callus in a stress-resistant inducing hormone-containing medium in advance before infecting Agrobacterium. It is presumed that the resistance to stress at the time of infection can be increased by previously culturing callus in a medium containing a stress resistance-inducing hormone. As the stress resistance-inducing hormone, any hormone known as a plant hormone having a stress relieving action may be used, and examples thereof include abscisic acid (ABA) and jasmonic acid. The stress resistance-inducing hormone used in the present invention is preferably abscisic acid.

  The period of culturing in the stress-resistant inducing hormone-containing medium may be the entire period of the culture period after callus induction or a part of the period. In particular, it is preferable to culture in a stress-resistant inducing hormone-containing medium for a period immediately before Agrobacterium infection. Specifically, the cells are cultured for 2 to 10 days immediately before the infection in a medium in which a stress resistance-inducing hormone is added to a callus induction medium. The stress-resistance-inducing hormone concentration in the medium is not particularly limited as long as it has an effect of increasing the gene transfer efficiency. The type of stress-resistance-inducing hormone to be added, the culture period, and the callus-inducing medium It can be appropriately determined in consideration of the type, the type of plant tissue used as a raw material for callus induction, and the like. For example, when ABA is used as the stress resistance-inducing hormone, it can be added to the callus induction medium so that the final concentration is 0.05 to 5 μM, preferably 0.1 to 2.5 μM.

  In addition, it is also preferable to grow transformed cells (callus infected with Agrobacterium) obtained after infection with Agrobacterium in a stress-resistant inducing hormone-containing medium. This is because by exposing (contacting) the stress-resistance-inducing hormone after infection, the effect of expressing the introduced target gene or the like in the transformed cell can be reduced. In particular, when a gene or the like having a large influence on the physiological function of a cell is introduced as a target gene, the callus infected with Agrobacterium is grown in a medium containing a stress-resistance-inducing hormone. The introduction efficiency can be remarkably improved.

  Specifically, the callus after being immersed in the suspension of the genus Agrobacterium is planted and grown in a medium in which a stress resistance-inducing hormone is added to a plant cell culture medium. The stress resistance-inducing hormone to be added, the concentration thereof, and the like are almost the same as when added to the callus induction medium. Note that the period of culturing in the medium containing the stress resistance-inducing hormone may be the entire period of the growth period after infection or a part of the period.

  Thus, the method for producing a transformed plant of the present invention is a method that can significantly improve the gene transfer efficiency by optimizing both the infectivity of the genus Agrobacterium and the culture state of the callus. is there. Latex-producing plants such as Para rubber tree have poor gene transfer efficiency, and the conventional method can transfer genes only about 10% of the total callus, but the method for producing the transformed plant of the present invention As a result, it is possible to achieve a very high efficiency of about 50% or more for gene transfer, and stably produce transformed cells.

<Method for creating transformed plant>
The method for producing a transformed plant of the present invention comprises inducing a somatic somatic embryo from the transformed cell produced by the method for producing a transformed cell of the present invention, and regenerating the plant from the somatic somatic embryo. Features. Induction of somatic embryos from transformed cells and regeneration of plant bodies from somatic embryos can be performed by appropriately adjusting techniques used in the field of plant engineering. For example, after transforming plant cells are cultured using a hormone-free regeneration medium or the like to induce somatic somatic embryos, the somatic embryos are cultured in a growth medium to form stems and roots. Thus, a plantlet can be obtained. A transformed plant can be obtained by transplanting and planting this plantlet in soil or the like.

  As a regeneration medium, a growth medium, etc., in a latex-producing plant such as para rubber tree, a medium used for the formation of somatic somatic embryos or regeneration of a plant can be used as it is or appropriately modified (for example, (See Patent Document 2). For example, as a regeneration medium, MS medium, MB medium, MS modified medium (medium in which the composition of the MS medium is changed), MB modified medium, or WPM medium are combined with cytokinin plant hormone, auxin plant hormone, gibberellin. A medium supplemented with hormones such as abscisic acid can be used. Further, the regeneration medium may further contain sugars such as sucrose and maltose, polyamines such as spermidine, nutrient sources such as coconut water, banana powder, and casein. The regeneration medium may be a liquid medium, but is preferably a medium using gellite, agar or the like as a callus support. On the other hand, the growth medium includes MS medium, MB medium, MS modified medium (medium in which the composition of the MS medium is changed), MB modified medium, or WPM medium, cytokinin plant hormone, auxin plant hormone, gibberellin, etc. A medium supplemented with the above hormones can be used. Furthermore, sugars such as sucrose and maltose, activated carbon and the like may be added. The growth medium may be a liquid medium, but is preferably a medium using gellite, agar or the like as a callus support. In addition, as for the density | concentration of each hormone, the density | concentration similar to the said callus induction medium is mentioned.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Reference Example 1]
<Growth curve of Agrobacterium>
The growth of Agrobacterium tumefaciens EHA105 strain, which is widely used in Agrobacterium, was confirmed.
First, Agrobacterium tumefaciens EHA105 strain (hereinafter referred to as EHA105 strain) is placed in 10 mL of LB medium in an L-shaped tube so that the initial bacterial count becomes 1 × 10 ⁶ to 1 × 10 ² cells / mL. Inoculated and inoculated, and cultured with shaking at 28 ° C. and 50 rpm (rotation / min). About 8 hours, the absorbance (OD ₆₆₀ ) of each medium with respect to light having a wavelength of 660 nm was measured to prepare a growth curve.

FIG. 1 is a graph showing the absorbance (OD ₆₆₀ ) measured at each culture time. From the left, the five curves in the figure show that the number of initial bacteria is 1 × 10 ⁶ cell / mL, 1 × 10 ⁵ cell / mL, 1 × 10 ⁴ cell / mL, 1 × 10 ³ cell / mL, 1 × It is a growth curve in the case of 10 ² cells / mL. Thus, it was found that by setting the initial bacterial count to 1 × 10 ⁴ cells / mL, the middle phase of the logarithmic growth phase was reached after 24 hours of culture. The middle logarithmic growth phase means a state where the growth curve shown in FIG. 1 can be approximated by a straight line and the OD ₆₆₀ is in the range of about 0.4 to 1.3.

[Example 1]
<Preparation of target gene-containing Agrobacterium>
A GFP gene encoding GFP that is a fluorescent protein and an nptII gene that is a neomycin resistance gene were used as target genes.
First, a GFP gene-containing binary vector into which the GFP gene and the nptII gene were incorporated was prepared and introduced into the EHA105 strain to prepare a GFP gene-containing EHA105 strain.
GFP gene-containing EHA105 strain cultured on antibiotic-added (50 mg / L kanamycin and 100 mg / L hygromycin) LB agar medium is scraped with a platinum loop so that the initial bacterial count becomes 1 × 10 ⁴ cells / mL. The LB liquid medium supplemented with antibiotics was inoculated and cultured with shaking at 28 ° C. under the condition of 50 rpm (rotation / min) for 24 hours to obtain cells in the middle of the logarithmic growth phase. The resulting _culture, those were diluted to _{OD 550} is 0.25 (approximately ^{5 × 10 8 cell / mL)} , and the GFP gene-containing strain EHA105 suspension.

<Preparation of callus>
Male flower buds used as explants were collected from para rubber tree, disinfected with diluted sodium hypozincate solution, then opened and the buds were taken out. This cocoon was planted in a callus induction medium (callus induction) and cultured in the dark at 27 to 28 ° C. for 5 to 12 weeks. As the callus induction medium, 1 to 3 mg / L BAP or KIN, 0.3 to 1 mg / L 2,4-D, and further 1 mg / L NAA or IAA are added to the MS basic medium, A medium supplemented with 7% sucrose and about 5% coconut water was used. The pH was adjusted to 5.6 to 5.8, and gellite or agar powder was used as the support.

<Infection>
The callus obtained by culturing for a predetermined period after callus induction was immersed in the GFP gene-containing EHA105 strain suspension prepared above for 1 minute, and then the callus was removed from the callus induction medium by removing hormones, and 20 mg / L It moved to the culture medium which added acetosyringone, and culture | cultivated in the dark at 22 degreeC for 5 days.

<Proliferation of callus containing transformed cells>
Further, the above callus was transferred to a medium containing 500 mg / L timentin after removing hormones from the callus induction medium, and cultured in the dark at 27 ° C. for 51 days (8 weeks after infection). Every 3 weeks, they were planted in the same medium.
The number of transformed cells in the obtained callus was measured by observing the fluorescence emitted by GFP, and the gene transfer efficiency was calculated. The calculated results are shown in Table 1. As is clear from these results, a high gene transfer efficiency of 50% or more is obtained by infecting callus obtained by culturing for 5 to 9 weeks after callus induction with Agrobacterium in the logarithmic growth phase. Can be achieved.

[Example 2]
<Preparation of target gene-containing Agrobacterium by conventional methods>
The GFP gene-containing EHA105 strain prepared in Example 1 was cultured on an LB agar medium supplemented with antibiotics (50 mg / L kanamycin and 100 mg / L hygromycin). The cells were cultured overnight in an added LB liquid medium. The obtained culture was diluted so that OD ₅₅₀ was 0.25 (about 5 × 10 ⁸ cells / mL), and was used as a GFP gene-containing EHA105 strain suspension (control).

<Creation of transformed cells>
The callus obtained by culturing for 8 weeks after callus induction in the same manner as in Example 1 was obtained by using the GFP gene-containing EHA105 strain suspension prepared in Example 1 or the GFP gene-containing EHA105 strain suspension prepared above. (Control) was immersed for 1 minute, and then infected and cultured in the same manner as in Example 1 to obtain callus for 8 weeks after infection.
By observing the fluorescence emitted by GFP, the ratio of the callus (GFP fluorescence callus; transformed cell) emitting fluorescence of GFP in the obtained callus was measured. The results are shown in Table 2. When the GFP gene-containing EHA105 strain suspension (control) prepared by the conventional method (samples 2A to 2C) was used, none of the GFP fluorescent callus reached 10% of the whole, and the GFP fluorescent callus No growth was observed. On the other hand, when the GFP gene-containing EHA105 strain suspension prepared in Example 1 was used (samples 2D to 2E), 50% or more of the total was GFP fluorescent callus, and GFP fluorescent callus proliferated. It was observed that In addition, the ratio which occupies for the whole GFP fluorescent callus means the same thing as gene transfer efficiency.
That is, from these results, even when callus obtained by culturing for 5 to 9 weeks after callus induction was used for infection, bacteria in the logarithmic growth phase were used as Agrobacterium spp. If not, it is clear that sufficient gene transfer efficiency cannot be achieved.

[Example 3]
Transgenic cell-containing callus obtained after infection was examined for gene transfer efficiency when cultured in a humidity-controlled environment.
First, in the same manner as in Example 1, the callus obtained by culturing for 8 weeks after callus induction was immersed in the GFP gene-containing EHA105 strain suspension prepared in Example 1 for 1 minute, and then the callus induction was performed. Hormones were removed from the medium, transferred to a medium supplemented with 20 mg / L acetosyringone, and cultured at 22 ° C. for 5 days in the dark.
Furthermore, the callus was divided into two, the hormone was removed from the callus induction medium, and the callus was transferred to a medium supplemented with 500 mg / L of timentin. One cell culture container sealed the lid | cover and the main body with the parafilm (sample 3A). The other cell culture vessel was sealed with a surgical tape (Micropore Surgical Tape, manufactured by 3M Company) having an average pore size of 0.1 μm or less (Sample 3B). Both cell culture vessels were placed in a constant temperature culture apparatus at 27 ° C. controlled to 60% humidity, and cultured for 51 days (8 weeks after infection) while being subcultured at intervals of 3 weeks.

  The number of transformed cells in the obtained callus was measured by observing the fluorescence emitted by GFP, and the gene transfer efficiency was calculated. The calculated results are shown in Table 3. Compared to the sample 3A cultured in a sealed environment with no humidity control, the sample 3B cultured in an environment adjusted to a humidity of 60% had a very high gene transfer efficiency of 80% or more. . From these results, it is apparent that the efficiency of gene transfer is further improved by culturing transformed cells in an environment in which humidity is controlled.

[Reference Example 2]
<Preparation 2 of target gene-containing Agrobacterium>
A GFP-HGGT chimera gene in which an HGGT gene encoding HGGT (homogenate gerenyl transferase transfer) protein was bound to the C-terminal of the GFP gene encoding GFP, and an nptII gene that is a neomycin resistance gene were used as target genes.
First, a GFP-HGGT gene-containing binary vector into which a GFP-HGGT chimera gene and an nptII gene were incorporated was introduced into the EHA105 strain to prepare a target gene-containing EHA105 strain. From the obtained GFP-HGGT gene-containing EHA105 strain, a GFP-HGGT gene-containing EHA105 strain suspension was prepared in the same manner as in Example 1.

[Example 4]
Before infecting with Agrobacterium, the gene transfer efficiency was examined when the callus was grown in a medium containing abscisic acid (ABA).
First, in the same manner as in Example 1, callus was induced from cocoons collected from Para rubber tree and cultured in callus induction medium for 8 weeks. The last one week was cultured in an ABA-containing medium obtained by adding ABA (0.5 μM) to the callus induction medium (sample 4B). What was cultured in the callus induction medium without addition of ABA for the last one week was used as a control (sample 4A).
The obtained callus was immersed in the GFP-HGGT gene-containing EHA105 strain suspension prepared in Reference Example 1 for 1 minute, and then infected and cultured in the same manner as in Example 1. Obtained.
By observing the fluorescence emitted by GFP, the ratio of the callus (GFP fluorescence callus; transformed cell) emitting fluorescence of GFP in the obtained callus was measured. The results are shown in Table 4. Unlike the case where GFP, which has a relatively small influence on the living body, was expressed alone, the gene transfer efficiency was as low as less than 20% when HGGT protein was expressed (Sample 4A). In contrast, in sample 4B cultured in an ABA-containing medium before infection with Agrobacterium, the gene transfer efficiency was very good at 46% . From these results, it is clear that the gene transfer efficiency is further improved by previously culturing callus in a stress resistant inducing hormone-containing medium for a predetermined period or more before infection with Agrobacterium.

  The method for producing transformed cells of latex-producing plants can improve the gene transfer efficiency when the Agrobacterium method is used, and is particularly useful in the field of natural rubber production.

Claims (9)

A method for producing a transformed cell of a latex-producing plant, comprising:
It has an infection process of infecting callus obtained by culturing latex-derived plant-derived tissue for 5 to 9 weeks after induction of callus with a bacterium belonging to the genus Agrobacterium containing a plasmid containing the target gene or a fragment thereof. ,
A method for producing a transformed cell of a latex-producing plant, wherein the Agrobacterium is a logarithmically growing bacterium. Furthermore, the callus infected with Agrobacterium in the infection step has a growth step of growing in a cell culture container,
The method for producing a transformed cell of a latex-producing plant according to claim 1, wherein the humidity in the cell culture container is adjusted to 50 to 70%.   The cell culture container sealed with a sealing member having a bacteria-impermeable and moisture-permeable property is installed in a constant temperature culture apparatus whose humidity is adjusted to 50 to 70%. Method for producing transformed cells of latex-producing plants.   Before the callus obtained by culturing for 5 to 9 weeks after the callus induction used in the infection step is infected with Agrobacterium, all or part of the culture period is contained in a medium containing stress-resistance-inducing hormone. 4. The method for producing a transformed cell of a latex-producing plant according to any one of claims 1 to 3, wherein the cell is cultured in a cell.   The latex-producing plant according to any one of claims 2 to 4, wherein in the growth step, callus infected with Agrobacterium is grown in a stress-resistance-inducing hormone-containing medium. How to make a cell.   The method for producing a transformed cell of a latex-producing plant according to claim 4 or 5, wherein the stress-resistance-inducing hormone is abscisic acid.   The method for producing a transformed cell of a latex-producing plant according to any one of claims 1 to 6, wherein the latex-producing plant is Hevea brasiliensis.   A somatic somatic embryo is induced from a transformed cell obtained by the method for producing a transformed cell of a latex-producing plant according to any one of claims 1 to 7, and the plant body is regenerated from the somatic embryo. A method for producing a transformed plant, characterized in that   A transformed plant of Hevea brasiliensis produced by the method for producing a transformed plant according to claim 8.

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