JP4374236B2 - Transformant using rubber tree culture system and evaluation method thereof - Google Patents

Description

  The present invention belongs to the field of genetically modified breeding of rubber tree and its evaluation technology.

  Natural rubber is manufactured from latex called rubber latex. As rubber tree cultivated for the purpose of industrial rubber raw material, the rubber tree of the family Euphorbiaceae, Havea brasiliensis, is particularly important, but as rubber tree that produces latex, there are other rubber tree (Manihot glaziovii), mulberry plant Indian rubber tree (Ficus elastica).

  It takes 5 to 7 years from planting rubber tree seedlings to growing to the extent that latex can be collected, and the collection period is approximately 20-30 years.

  In addition, about 35% of the rubber component in the latex produced by Para rubber tree, there are many useful substances such as protein, inositol, saccharides such as ketebraquitol, a kind of vitamin E, and tocotrienol which is also effective as a natural anti-aging agent. (Non-patent Document 1).

  In addition, for herbs, research on the production of genetically modified plants is progressing, but for trees, it is difficult to redifferentiate from cells, and it takes too long to obtain a plant, To date, research has been extremely limited.

  Some rubber trees are also being tried for genetically modified breeding, but it takes 3 months to regenerate into embryoid bodies, 60 more days to reach plantlets, and then check whether the gene is expressed in the plants. Therefore, it takes considerable time for breeding. (Patent Document 1)

Japanese Patent No. 3289021 Applied Rubber Chemistry 12 Lecture by Hideo Kaneko 52-53 Taiseisha

  In order to increase the production of useful substances derived from latex or other rubber trees, and to improve the environmental adaptability of rubber trees, it is desired to further improve the characteristics and properties of rubber trees.

  In order to improve the traits of rubber trees such as para rubber by gene introduction, the gene is introduced into callus derived from pods, immature seeds or formation layers, the callus is redifferentiated, the plant is regenerated, The effect will be evaluated. It takes a very long time to regenerate callus and regenerate a plant. Therefore, the evaluation is very delayed, and it takes a lot of time to repeat improvement and breed.

  Therefore, the object of the present invention is to breed a genetically modified plant having a desired characteristic in a short period of time by breeding a genetically modified plant by evaluating the recombinant plant as early as possible.

  The present inventors have found a condition for introducing a target gene into callus derived from rubber tree pods, immature seeds, or a formation layer, and then efficiently regenerating adventitious roots from callus. Then, the present invention has been completed by finding that by culturing genetically modified rubber tree adventitious roots, the function of the transgene in the plant can be evaluated and tested at the adventitious root stage.

  According to the present invention, by culturing genetically modified rubber tree adventitious roots, it becomes possible to evaluate genetically modified rubber tree at the stage of adventitious roots, so that it is possible to greatly reduce the breeding period of rubber tree plants by genetic modification technology, a new Thus, genetically modified rubber trees having the desired characteristics can be bred efficiently.

  In particular, by transformation, (1) by controlling the polymerization reaction of rubber, by changing the molecular weight or molecular weight distribution of the rubber in the latex, (2) by increasing the rubber biosynthesis reaction, by increasing the rubber weight of the rubber Or (3) When the synthesis of useful components is promoted to increase the alcohol, tocotrienol, and useful proteins, whether or not breeding as intended has been achieved at the adventitious root stage (1 It can be screened by molecular weight analysis, (2) quantification of rubber amount, (3) quantification of each component (liquid chromatography), etc., and can improve breeding efficiency.

  The invention of the present application introduces a gene into a callus or immature embryo derived from a moth, immature seed, or formation layer of rubber tree, then differentiates the callus or immature embryo into an adventitious root, and cultures the adventitious root of the genetically modified rubber tree. Of genetically modified adventitious roots.

  Furthermore, the present invention also includes a method for evaluating and assaying transformation by a target gene introduced at the adventitious root stage, using adventitious roots modified with rubber tree.

  Particularly preferred examples of the rubber tree to be subjected to tissue culture include Para rubber tree, but besides this, Cela rubber tree, Indian rubber tree, Panama rubber tree, guayule rubber tree, rubber dandelion, Lagos rubber tree, gutta pergano tree, parata rubber tree, cis Chicle.

  Hereinafter, para rubber trees will be described as an example, but other rubber trees can be implemented by the same method.

  As materials used for tissue culture, various parts such as para rubber tree roots, stems, leaves or flowers can be cut into appropriate sizes, but preferably buds, immature fruits or stems are used. The formation layer taken out from the bud taken out from the bud or from the immature seed taken from the immature fruit or the stem can be used as the material.

  First, the surface of the para rubber tree material is washed. When using the inside of a plant as a material, for example, it may be washed with a scouring powder, but it can also be washed with water containing about 0.1% of a surfactant. When using leaves, wash the surface with a soft sponge.

  The material is then sterilized or sterilized. Sterilization or sterilization can be performed using a well-known disinfectant / sterilant, and ethanol, benzaconium hydrochloride, and sodium hypochlorite aqueous solution are preferable.

  Callus can be derived from material taken from para rubber tree, adventitious roots can be derived from callus, or sterile roots can be derived directly from material collected from para rubber.

I. Callus induction method Preferably, cocoons, immature seeds or formed layers are used as the material. To induce callus, cytokinin plant hormones and / or auxin plant hormones are added to the medium at a low concentration. Medium includes 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, etc. However, MS medium or its modified or MS modified medium 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 ppm.

  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 ppm of 2,4-D (dichlorophenoxyacetic acid) and about 1 ppm of IAA (indoleacetic acid) or about 1 ppm 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 can be used in combination.

  Furthermore, in order to promote callus induction, sucrose may be added at a high concentration of 6 to 7 wt%, and coconut water may be added. Preferably, the pH is adjusted to 5.6 to 5.8, the culture temperature is 26 to 28 ° C., and callus is induced in the dark. As the callus support, gellite or agar can be used.

  The induced callus is cultured in a dark medium at the same composition for 1 to 2 months at a culture temperature of 26 to 28 ° C. to grow the callus. Suitable callus is obtained in 6-7 weeks.

II. Preparation method of immature embryo Preferably, an immature green fruit at a stage before the outer pericarp is hardened is used. The surface of the immature fruit is sterilized with well-known sterilizing agents such as ethanol, benzaconium hydrochloride, sodium hypochlorite, preferably ethanol. After sterilization, the seeds inside the fruit are removed. The part that grows into an immature seed embryo is removed.

  The extracted immature embryo is grown in a medium. Examples of the medium include a White medium, a Heller medium, an SH medium, an MS medium, an LS medium, and the like, and an MS medium or an MS modified medium obtained by changing the composition thereof is preferable.

III. Gene transfer method
III-1 Preparation of target gene recombination vector Target genes to be introduced into rubber trees include, for example, genes involved in the biosynthesis mechanism of rubber and polyisoprene chain extension reaction, which function on latex production and molecular weight, and A protein that is involved in the biosynthesis of tocotrienol, which is a kind of vitamin E, which is a kind of saccharides such as protein, inositol, and quebrakitol, and is also effective as a natural anti-aging agent, contained in latex, In addition, genes that produce mutants of these proteins, saccharides, and tocotrienols can be mentioned, but the present invention is not limited to these, and any gene can be introduced. The target gene is placed on a vector linked to a promoter that functions in rubber tree. As a promoter that functions in rubber tree, it can be used as long as it functions in rubber tree, whether it is a promoter derived from rubber tree or from a different species. For example, CaMV35, NOS promoter, etc. Can be used.

  The target gene is preferably incorporated into the vector together with a marker gene and optionally a reporter gene.

  Examples of the selection marker include kanamycin resistance gene (nptII), hygromycin resistance gene (hptI), bleomycin resistance gene and the like. Examples of reporter genes for confirming the expression position in the plant include luciferase and GUS (β-glucuronidase) gene.

III-2 Method of introducing target gene into callus or immature embryo Target recombinant vector is introduced into callus or immature embryo by well-known means such as electroporation method, Agrobacterium method, particle gun method, etc. .

III-2-1 When using the particle gun method, proceed as follows. First, gold particles (preferably having a diameter of 1-2 μm) are coated with a target gene recombination vector and adhered to a microcarrier. Callus or immature embryos are arranged on a plate and driven twice with a particle gun device.

III-2-2 The Agrobacterium method is as follows.
The target gene recombination intermediate vector prepared in III-1 above is homologously recombined with the Ti plasmid in Agrobacterium tumefaciens, or the target gene binary vector is introduced into Agrobacterium tumefaciens.
The target gene can be introduced into the callus by infecting the callus with the transformed Agrobacterium.

III-3 Electroporation method
Protoplasts are prepared from the callus prepared in III-1. In the presence of the target gene recombination vector prepared in III-1, the prepared protoplast is subjected to a high piezoelectric field in the solution to introduce the target gene into the protoplast.

IV. Method for Inducing Adventitious Roots from Callus Transgenic callus or protoplasts are grown in a medium.
Adventitious roots are induced from the grown callus. For the induction of adventitious roots, a medium in which 3 to 10 ppm of IBA (indolebutyric acid) is added to the MS basic medium is preferably used. More preferably, 6-7 wt% of sucrose is added. Auxins such as IAA and NAA may or may not be added.

V. Induction of sterile roots from immature embryos Transgenic immature embryos are grown in medium. Examples of the medium include a White medium, a Heller medium, an SH medium, an MS medium, an LS medium, and the like, and an MS medium or an MS modified medium obtained by changing the composition thereof is preferable.

  If immature embryos cultured for about 30 days and relatively grown are added with sucrose on the basis of the MS basic medium, roots can be induced without much influence by the hormone species.

VII. Mass growth of roots Or 2. The adventitious or sterile roots induced in step 1 are cut into small pieces having an appropriate size, preferably 2 to 3 cm square. An appropriate medium, for example, White medium, Heller medium, SH medium, MS medium, LS medium, or MB medium can be used, and preferably MS medium is used, and IBA 3 to 10 ppm is added to the medium. By culturing, roots can be propagated. The medium may be solid or liquid. When using a solid medium, when a root grows to an appropriate size, a part of the root is cut and further transplanted to proliferate. Preferably, shake tow culture is performed using a liquid medium and a jar fermenter or the like. By culturing in liquid, more efficient growth is possible and the transplantation operation is facilitated.

VIII. Confirmation of target gene expression from roots A target gene product is extracted from a large-scale cultured and grown root culture.

  Furthermore, a trace amount extract can analyze a component by analyzing by methods, such as pyrolysis GC / MS, FT-IR, or NMR.

  Also, latex is extracted from a large amount of grown roots, and rubber components are separated by a technique such as centrifugation, and then useful substances such as proteins, sugars, and tocotrienols, which are products of target genes, are detected.

[Example 1] Induction of callus Latex production method by tissue culture (1) Method of obtaining adventitious roots from calli, immature seeds, and formation layers via callus Male flower buds used as explants are in a cocoon state It was collected from para rubber tree, disinfected with a diluted sodium hypozinc acid solution, and then opened and taken out. Immature seeds were collected from soft fruits with a green outer skin. In the formation layer, young branches were collected, and the surface was washed with ethanol and then sampled.

  For callus induction, add 1 to 3 ppm of BAP or KIN, 0.3 to 1 ppm of 2,4-D, and 1 ppm of NAA or IAA to the MS basic medium, and then add 6 to 7% sucrose and about 5% coconut. The medium to which water was added was prepared. The pH was adjusted to 5.6 to 5.8, and gellite or agar powder was used as the support.

Growth of callus was confirmed by culturing for 1-2 months in the dark at 27-28 ° C.
The grown callus was transplanted to a medium having the same composition as the callus induction, in which only the hormone composition was changed to 3 to 10 ppm. Auxins such as IAA and NAA may or may not be added. Adventitious roots could be induced by adding IBA and culturing for 1 to several months in an environment of 27 to 28 ° C. irradiated with light for 16 hours. The formed root is shown in FIG.

(2) Method for inducing sterile roots from immature embryos By sampling relatively grown immature embryos from seeds and culturing them in a medium containing 6-7% sucrose in an MS basic medium, there is little effect on hormone species. Without being able to induce sterile roots.

(3) Mass growth of roots The roots obtained in the cultures (1) and (2) were cut into small pieces of 2 to 3 cm and the roots were grown in a medium supplemented with 3 to 10 ppm of IBA. Good results were obtained with either solid or liquid medium. (See Figure 2.)

(4) <Confirmation of latex in cultured roots>
In the grown root culture, latex exudation was implicitly confirmed by cutting during transplantation. In order to further analyze the composition, Soxhlet extraction was performed on the cultured roots with an organic solvent (such as chloroform), and the extracted portion was analyzed by techniques such as pyrolysis GC / MS, FT-IR, and NMR.
For example, when CDC13 was measured by NMR using a solvent as a solvent, high peaks were observed at positions of 135.24 ppm, 125.08 ppm, 32.25 ppm, 26.44 ppm, and 23.44 ppm. In addition, 128.0 ppm, 120 Low peaks were observed at positions of .4 ppm, 29.7 ppm, 22.7 ppm and 14.1 ppm.

  As a result, it was confirmed that cis polyisoprene, which is a main component of natural rubber, was present in large quantities, and useful components such as sugars, amino acids, proteins, and higher fatty acids were identified. (See Fig. 3 NMR spectrum)

[Example 2] Gene introduction into callus (1) Preparation of plasmid to be introduced into callus Here, β-glucuronidase (common name: GUS) is used as a reporter protein. GUS is an enzyme that acts on the β-type glycoside of D-glucuronic acid to hydrolyze its glucuronide bond, and does not exist in rubber tree. Therefore, a plasmid having a GUS gene and an antibiotic resistance gene for selecting colony was designed as follows.

  A GUS gene and a hygromycin resistance gene (hygr) are incorporated into the multicloning site of pUC19 so as to be downstream of the promoter of CaMV35S to prepare pU19-GUS.

(2) Introduction of plasmid into callus A particle gun (BIORAD PDS 1000 / He) is used for gene introduction. The rupture disk uses 1100 psi (7.58 Mpa). The distance from the micro carry to the stopping screen is 1.0 cm, and the distance from the stopping screen to the callus sample to be driven is 9.5 cm. For the shot, 1.6 μm gold particles coated with the above-mentioned pCU19-GUS plasmid are used.

(3) Primary selection Callus is transferred to a selection medium containing hygromycin two days after gene transfer, cultured, and the callus that has grown is primarily selected.

(4) Induction of root The gene-introduced callus or protoplast is grown in a medium.
Adventitious roots are induced from the grown callus. For the induction of adventitious roots, a medium in which 3 to 10 ppm of IBA (indolebutyric acid) is added to the MS basic medium is preferably used. More preferably, 6-7 wt% of sucrose is added.

(5) Mass cultivation of roots The adventitious roots induced in (4) above are cut into small pieces of about 1 cm square. Root growth can be performed by adding 3-10 ppm of IBA in MS medium and culturing using a jar fermenter.

(6) Confirmation of GUS expression Mass tissue (0.1 g) grown in large quantities was collected, and GUS extraction buffer (50 mM sodium phosphate buffer, 10 mM EDTA, 0.1% Triton X-100, 0.1% sarkosyl, 10 mM 2-ME) Add 0.5 ml) and homogenize well. Transfer the entire volume to an Eppendorf tube and centrifuge for 5 minutes. The supernatant is transferred to a new Eppendorf tube, from which 10 μl is taken and transferred to another Eppendorf full tube, which is used as a GUS-containing sample. Next, GUS substrate solution (1 mM 4-MUG (4-methyl-umbelliferyl-β-D-glucuronide, 50 mM sodium phosphate buffer, 10 mM EDTA, 0.1% Triton X-100, 0.1% sarkosyl, 10 mM) 2-ME) Add 90μl and incubate at 37 ℃ for 1 hour, then add 900μl of reaction stop solution (0.2% Na2CO3) to stop the reaction.Quantify fluorescence intensity with a fluorescence spectrophotometer (excitation 365nm, fluorescence 455nm). Measure automatically.
As a result, it is possible to screen the gene-introduced root.

Callus rooting in medium containing IBA Elongated cultured root 13C-NMR spectra of extracts extracted from cultured roots with chloroform

Claims (4)

Rubber tree anther, by introducing a target gene into callus derived from immature seeds or forming layer, from the callus using a medium supplemented with 3~10ppm IBA (indole butyric acid) and 6～7Wt% sucrose, adventitious roots or sterile roots A method for cultivating transformed roots of rubber tree, which comprises cultivating the roots into small pieces and culturing them in a medium containing IBA. A target gene is introduced into a tissue culture of immature embryos of rubber tree, and adventitious or sterile roots are induced from the tissue culture using a medium supplemented with 6 to 7 wt% sucrose, and the roots are cut into small pieces containing IBA A method for cultivating transformed roots of rubber tree, comprising culturing in a culture medium. The method for cultivating transformed roots of rubber tree according to claim 1 or 2 , wherein the rubber tree is para rubber tree. The target gene is a gene involved in the biosynthesis mechanism of rubber or polyisoprene chain extension reaction, or a gene involved in the biosynthesis of protein, saccharides such as inositol and quebrachitol, or tocotrienol contained in latex. 1-3 . A method for cultivating a transformed root of rubber tree according to any one of claims 1 to 3 .

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