JP4271598B2 - Tomato cell line, production method thereof, and culture medium for tomato cell line - Google Patents

Description

  The present invention relates to a tomato cell line that can be continuously subcultured, a production method thereof, and a culture medium for tomato cell line culture.

  Conventionally, the Tm-1 trait has been known as a trait that suppresses the growth of tomato mosaic virus (ToMV, hereinafter abbreviated as ToMV; one kind of tobamovirus) in tomato. The Tm-1 trait originates from wild tomato species (Lycopersicon hirsutum) and is widely used for cultivated species for breeding, but until the Tm-1 gene is cloned and the base / amino acid sequence is specified. Not reached.

  The Tm-1 trait is a semi-dominant trait, and the homozygote (Tm-1 / Tm-1) has a greater degree of suppression of ToMV growth than the heterozygote (Tm-1 / +). However, the mechanism of ToMV growth inhibition is not well understood.

  In order to perform such functional analysis of Tm-1 trait (for example, Tm-1 gene cloning, identification of base sequence / amino acid sequence, analysis of ToMv growth inhibitory mechanism, etc.) It is essential to establish a cell line (cell line) having a Tm-1-like function.

For example, in Non-Patent Document 1, callus induction is performed on a Tm-1 tomato cell line, and subculture of the grown callus is performed. Specifically, in Non-Patent Document 1, B5 medium supplemented with kinetine sucrose and yeast extract, homozygote having Tm-1 trait, and near-isogenic and not having Tm-1 trait Cell lines and cell lines derived from heterozygotes obtained by crossing the two are cultured, and the grown callus is subcultured.
Watanabe, Y., Kishibayashi, N., Motoyoshi, F. and Okada, Y. (1987) Characterization of Tm-1 gene action on replication of common isolates and a resistance-breaking isolate of TMV. Virology 161: 527-532.

  However, the cell line obtained by the technique of Non-Patent Document 1 has a problem that it is browned and unstable, and the growth efficiency is low. In fact, the Tm-1 trait homozygote, heterozygote, and cell line not having the Tm-1 trait have already disappeared.

  Thus, it is difficult to establish a tomato cell line by the conventional culture technique. For this reason, a stable and highly efficient tomato cell line cannot be established, and a uniform cell line cannot be produced in large quantities. Therefore, it is highly desired to establish a tomato cell line that is stable and has high growth efficiency.

  The present invention has been made in view of the above-described conventional problems, and the object thereof is tomato cell line that is stable and has high growth efficiency, a production method thereof, and culture and production of such a tomato cell line. It is to provide a suitable culture medium for tomato cell line.

  As a result of intensive studies in view of the above problems, the present inventors have found that by culturing tomato cells in a medium containing zeatin (a kind of cytokinin), a tomato cell line having high stability and proliferation efficiency can be produced. The present invention has been completed.

  That is, the production method of the tomato cell line of the present invention is characterized in that tomato cells are cultured in a medium containing zeatin in order to solve the above-described problems.

  The method for producing a tomato cell line of the present invention is characterized by culturing tomato cells in a medium containing zeatin and at least one compound of auxin, cytokinin, and saccharide in order to solve the above-mentioned problem. It is said.

In the production method of the tomato cell line of the present invention, it is preferable to perform shaking culture under a condition where the swing width is 3 cm or more.
In the production method of the tomato cell line of the present invention, it is preferable that the medium further contains α-naphthalene acetic acid, 2,4-dichlorophenoxyacetic acid, and sucrose.

  The tomato cell line of the present invention is obtained by the above-described production method of the tomato cell line of the present invention.

  The tomato cell line of the present invention may have a Tm-1-like character.

  The tomato cell line culture medium of the present invention is characterized by containing zeatin.

  The tomato cell line culture medium of the present invention is characterized by containing zeatin and at least one compound of auxin, cytokinin, and saccharide.

  The tomato cell line culture medium of the present invention may further contain α-naphthalene acetic acid, 2,4-dichlorophenoxyacetic acid, and sucrose.

  According to each of the above-described configurations and methods, it is possible to establish a tomato cell line that has been difficult in the past and does not brown, is stable, and has high growth efficiency. That is, a tomato cell line that can be continuously subcultured can be provided. Therefore, since a large amount of uniform tomato cell lines can be obtained, it is useful for various functional analysis of tomatoes and determination of traits (for example, determination of Tm-1 traits).

  As described above, the production method of the tomato cell line of the present invention is to culture tomato cells in a medium containing at least zeatin, and the medium for culturing tomato cell lines of the present invention contains at least zeatin. The tomato cell line of the present invention is cultured in a medium containing at least zeatin. Therefore, it is possible to produce a tomato cell line that is stable and has high growth efficiency, and to provide a uniform tomato cell line that can be continuously subcultured.

  Hereinafter, an embodiment of the present invention will be described, but the present invention is not limited thereto.

(1) Tomato culture medium First, the tomato culture medium of the present invention (hereinafter referred to as the present medium) will be described. This medium is a medium suitable for culturing tomatoes, and in particular, a medium that can be used to establish a tomato cell line that has been difficult in the past and has stable and high growth efficiency. That is, this medium is a medium particularly suitable for tomato tissue culture in which tomato cells, tissues, organs, etc. are taken out and cultured aseptically.

  A medium is a particularly important element in tissue culture. This is because the state of cell division and differentiation varies depending on the composition of the medium. The greatest feature of this medium is that it contains the plant hormone zeatin as an essential component.

  Because plant hormones are essential for root and leaf differentiation and growth, plant hormones are essential reagents in tissue culture. In addition to tissue culture, various plant hormones such as auxin root promoters that promote root growth during cutting, side-bud-induced benzyladenine preparations, and gibberellin that promotes seed germination, even in normal cultivation Is used. In particular, in order to induce cell lines to callus and adventitious buds and to grow plant individuals in large quantities, it is necessary to use plant hormones.

  In tissue culture, the types and amounts of various additives (medium components) including plant hormones have a great influence on cell division and differentiation of cell lines and the like. For example, as described above, the tomato cell line obtained by the culture method described in Non-Patent Document 1 is browned and unstable, and has a low growth efficiency. The browned cells are dead (or nearly dead) cells and cannot proliferate. That is, the greater the degree of browning, the worse the growth efficiency. The degree of browning (cell line state) can be visually confirmed with a microscope or the like.

  Thus, in order to establish a stable tomato cell line with high growth efficiency, it is necessary to fully study various additives, particularly plant hormones.

  Therefore, this medium is characterized by containing zeatin as a plant hormone. Zeatin is a kind of naturally occurring plant hormone (plant growth hormone), and more specifically, a kind of cytokinin that is a plant hormone that promotes cell division of plants.

  The amount of zeatin added is not particularly limited as long as the tomato cells are in a range in which they do not brown and stably grow. For example, zeatin can be added at a rate of 0.1 to 2.0 mg (general use amount of zeatin) per liter of medium. In Examples described later, 0.2 mg of zeatin is used per 1 L of the medium.

  Such various additives including zeatin may be added to a basic medium for plant cell culture. This basic medium may be any plant cells that can be cultured. Some basic media are widely used for cultivation of plants and plants, and a predetermined amount of an inorganic substance or the like is mixed in advance. The basic medium is not particularly limited. For example, Murashige-Suguog medium (MS medium), B5 medium (also called Gamborg B5 medium), Hyponex medium (H medium), Knudson C medium (KC medium) ), These modified media (for example, modification of the composition ratio, etc.) and the like.

  In other words, this medium contains plant hormones, inorganic salts, carbon sources, nitrogen sources, vitamins, minerals (generally used in plant culture media as long as they do not affect the stability and cell division of tomato cells. Various additives such as nutrients such as phosphorus, calcium, magnesium, iron, etc.), amino acids, peptone, sugars, growth promoters (coconut milk and yeast extract) may be added. These additives may be used alone or as a mixture.

  As plant hormones, for example, auxin (indole acetic acid and compounds showing an equivalent action; indole acetic acid, indole butyric acid, naphthalene acetic acid, 2,4-dichlorophenoxyacetic acid, etc.), cytokinin (kinetine and equivalent actions) And compounds such as kinetin, benzyladenine, etc.), gibberellin, abscisic acid, ethylene, jasmonic acid, thidiazuron (TDZ), brassinosteroid, florigen and the like. The plant hormones include not only naturally occurring natural plant hormones but also chemically synthesized synthetic plant hormones having the same action as natural plant hormones. In addition, the plant hormone is preferably one that promotes proliferation (growth) of tomato cells.

  In addition, as shown in the below-mentioned Example, this culture medium is a medium (NDZ medium) further containing α-naphthalene acetic acid and 2,4-dichlorophenoxyacetic acid as an additive other than zeatin, for example, as a plant hormone. It is preferable that a saccharide-added NDZ medium in which a saccharide such as sucrose is added to the NDZ medium is more preferable.

  Further, the present medium may be a solid medium or a liquid medium. This medium can be prepared by mixing the above-mentioned various additives (medium material), placing them in a culture vessel, adding water, stirring, and heat sterilizing. As the culture vessel, for example, a commonly used glass or polycarbonate transparent or translucent vessel that can withstand heat sterilization can be used, and is not particularly limited. Further, the heat sterilization time varies depending on the scale of culture and the type of sterilizer, and is not particularly limited.

  In addition, by making this culture medium into a kit, a tomato cell line can be easily established only by aseptically inoculating a part of the tomato into the kit.

  By using such a main medium, a stable and highly efficient tomato cell line can be produced, and a uniform tomato cell line that can be continuously subcultured can be provided.

(2) Tomato cell line of the present invention and production method thereof The production method (present production method) of the tomato cell line of the present invention comprises culturing tomato cells using the above-mentioned medium, and the tomato of the present invention A cell line is a tomato cell line which is obtained by this production method and does not brown.

  That is, this production method includes a step of aseptically culturing tomato cells in a tomato cell line culture medium.

  In this step, the culture conditions such as temperature, light, humidity, and culture period are not particularly limited because they vary depending on the type of tomato cell used and the character. As culture conditions, for example, stationary culture on a solid medium, shaking culture in a liquid medium, and the like can be applied. In addition, after culture | cultivating with a solid culture medium, you may combine culture | cultivation with a solid culture medium, such as culture | cultivation with a liquid culture medium.

  In shake culture, the medium container generally has a reciprocal swing width of about 2 cm. For this reason, this production method may be carried out under general shaking culture conditions. However, in this production method, it is preferable to perform shaking culture under conditions exceeding a general reciprocal swing width. For example, it is preferable that shaking is performed under the condition that the normal swing width is 3 cm or more (in the examples described later, the swing width is 7 cm that is three times the normal swing). Thereby, a more stable tomato cell line is obtained. If the swing width is too large, cultured cells adhere to the side wall of the container. For this reason, it is necessary to set an upper limit of the swing width so that the cultured cells do not adhere too much to the side wall.

  In this production method, the tomato cells to be cultured may be a part of a tomato individual (eg, stem, root, leaf, bud, etc.). Moreover, the tomato cell to be used may have any character. The characteristics of tomato cells are not particularly limited, but as in the examples described later, in particular, tomato cells having a Tm-1-like character (for example, the GCR237 (Tm-1 / Tm-1) line, or , Tm-1 / + line, etc.)), GCR237 and GCR26 (+ / +) line which is near-isogenic and does not have Tm-1 are preferred. That is, the character of the tomato cell (character of the tomato cell line) is not particularly limited. For example, a Tm-1-like character is preferable.

  Here, the “Tm-1-like trait” is a trait that a plant has and can suppress the proliferation of tobamovirus. Such tomato cell lines having Tm-1-like traits are useful for functional analysis of Tm-1-like traits whose details have not been clarified (analysis of gene sequences and amino acid sequences, analysis of viral growth suppression mechanisms, etc.) It is. The tobamovirus is a virus belonging to the genus Tobamovirus, and the genus Tobamovirus is a positive-stranded single-stranded RNA virus group classified into an alpha-like virus supergroup.

  According to this production method, it is possible to produce a tomato cell line that is stable without browning as in the prior art and has high growth efficiency. For this reason, this cell line can be continuously subcultured. In other words, a small amount of tomato cell line can be used to induce and share many calli, protoplasts, and the like. Thereby, mass growth of the same genotype tomato cell line and production of a uniform cell line are possible.

  The proliferation efficiency can be confirmed by monitoring cell proliferation. Examples of the method for monitoring cell growth include a method of counting the cell concentration with a microscope at regular intervals after cell transplantation, or a method of measuring the cell weight or cell volume after removing the medium. The Further, the stability of the cells can be judged by the color (colored state) when the cells are collected and the form of microscopic observation. Specifically, when a brown cell is observed with a microscope, it can be seen that the cell can hardly or not proliferate. Therefore, brown cells (colored cells) can be regarded as dead cells (cells that cannot proliferate). That is, cells that are not colored are more stable and have higher proliferation efficiency.

  As described above, according to this production method, it is possible to produce a tomato cell line which is stable without browning and has high growth efficiency. Moreover, many plant individuals can be obtained from a small amount of material by inducing or differentiating this tomato cell line.

  Moreover, since this production method performs tissue culture, there is an advantage that the cell line can be induced to a relatively uniform cell population (callus). In addition, since tissue culture can be adjusted with high precision, the production method can be applied to the analysis of the effects of temperature, light, humidity, various substances, etc. on plant growth and differentiation. . In addition, this production method using this medium can obtain many plant individuals from a small amount of plant material (plant cells and tissues), thus shortening the growing period of a new kind of tomato that is particularly useful in the agricultural field. The cell line can be preserved by subculture.

  In addition, a stable and highly efficient tomato cell line can be produced and a uniform cell line can be produced in large quantities, so that an experiment under the same conditions using the uniform cell line is possible.

  Moreover, since the presence or absence of a Tm-1-like character cannot be determined from appearance, a tomato cell line having a Tm-1 character can be used to determine the presence or absence of a Tm-1-like character in a wild-type tomato. It can also be used to analyze the base sequence and amino acid sequence of the Tm-1 gene and to elucidate the mechanism of inhibition of tobamovirus growth. Note that the present invention is not limited to the tomato cell line having the Tm-1 trait, and can be used for any application using a uniform and stable tomato cell line.

  Moreover, this production method and this culture medium can be applied not only to tomatoes but also to the solanaceous plants to which the tomatoes belong, and the same effects can be obtained.

  Thus, according to the present invention, it is possible to induce a callus (cell mass) and establish a stable cell line that can be differentiated into various tissues, organs, and individuals. Furthermore, it becomes possible to obtain a large number of proliferating cells that are difficult to obtain in higher plants. Further, the obtained cell line can be used as a stable experimental material. Moreover, it can apply also to the use which observes directly the influence on the cell line of the added substance by adding a substance to the culture medium which culture | cultivated the cell line. Thus, the present invention can be applied to various uses, and can be used in a wide range of fields from basic research to applied research.

  It should be noted that the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims, and the embodiments obtained by appropriately combining the respective technical means disclosed are also included in the present invention. It is included in the technical scope of the invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to a following example.

[Example 1] Production of tomato cell line using zeatin-containing medium (1) Production of cultured cells of tomato 0.2 mg / L of zeatin (Zeatin) as a plant hormone, and 2 mg of α-naphthalene acetic acid (α-NAA) / L, 2,4-Dichlorophenoxyacetic acid (2,4-D) 0.2 mg / L, Murashige & Sugug medium supplemented with 3% (w / v) sucrose (this medium is referred to as NDZ medium) Used for induction. First, aseptically sowed and cultivated two tomato (+ / +, Tm-1 / Tm-1) stem fragments were placed on an NDZ agar medium and statically cultured in a dark place at 23 ° C. The induced callus was suspended in NDZ liquid medium, shake-cultured (23 ° C., 70-80 rpm (reciprocating; swing width 7 cm)), tomato cell lines GCR26 (+ / +) and GCR237 (Tm−1 / Tm− 1) was established. All of the obtained cell lines were stable without browning and had high growth efficiency. GCR237 is a homozygote that repeats backcross to the tomato variety Craigella (Tm-1 / Tm-1), and GCR26 is a homozygote that repeats backcross to Craigella (+ / +) It is.

(2) Preparation of Protoplast from Tomato Cultured Cells The above GCR237 (Tm-1 / Tm-1) cultured cells were prepared in NDZ liquid medium at 23 ° C., 70-80 rpm (reciprocating; swing width 7 cm), in the dark, 7 The culture was shaken using a flask for one day. Thereby, cells in a steady state were obtained. The cells were further subcultured to fresh NDZ medium and cultured for 3 days under the same conditions. The supernatant was removed by centrifugation (LC-121, Tommy Seiko, 800 rpm, 1 minute), and the cells were collected. 0.7 M mannitol (30 ° C.) was added to the obtained cells, and the cells were allowed to stand at room temperature for 10 minutes. This was centrifuged again (Tomy Seiko LC-121, 800 rpm, 1 minute) and the supernatant was removed. Then, a protoplast preparation solution (1% cellulase “ONOZUKA” RS (manufactured by Yakult Co.), 0.2% pectinase, 5 mM CaCl ₂ , 0.6 M mannitol was dissolved in NDZ medium and the pH was adjusted to 5.8), and reacted at 30 ° C. for 2 hours to prepare protoplasts.

[Example 2]
The stems of two lines of tomato (+ / +, Tm-1 / Tm-1) were aseptically inoculated and cultured in the same manner as in Example 1 except that the shaking width of the shaking culture was 2 cm. The obtained cell line was slightly browned, but its growth efficiency was lower than that of Example 1.

[Comparative Example 1]
The composition of the medium was other than that described in Non-Patent Document 1, that is, it contained 0.2 mg / L of kinetin as a plant hormone, and further 5% (w / v) yeast extract and 3% sucrose (w / v ) Was added aseptically to the stems of two lines of tomato (+ / +, Tm-1 / Tm-1) and cultured as in Example 1 except that 0.9% agar was added. . The obtained cell line was immediately browned and the growth efficiency was low.

[Comparative Example 2]
Two lines of tomato (+ / +, Tm) were used in the same manner as in Example 1 except that the medium composition was a Murashige & Sugog medium containing 0.5 mg / mL 2,4-dichlorophenoxyacetic acid as a plant hormone. -1 / Tm-1) stems were aseptically inoculated and cultured. The obtained cell line was immediately browned and the growth efficiency was low.

  As described above, according to the present invention, a tomato cell line that is stable without browning and has high growth efficiency can be provided. Therefore, it is suitably used for functional analysis of various traits using the tomato cell line. be able to.

Claims (9)

  A method for producing a tomato cell line, comprising culturing tomato cells in a medium containing zeatin.   A method for producing a tomato cell line, comprising culturing tomato cells in a medium containing zeatin and at least one compound of auxin, cytokinin, and sugar.   The method for producing a tomato cell line according to claim 1 or 2, wherein shaking culture is performed under a condition where the swing width is 3 cm or more.   The method for producing a tomato cell line according to claim 1, wherein the medium contains α-naphthalene acetic acid, 2,4-dichlorophenoxyacetic acid, and sucrose.   The tomato cell line obtained by the production method of the tomato cell line of any one of Claims 1-4.   The tomato cell line according to claim 5, which has a Tm-1-like character.   A culture medium for tomato cell line culture, comprising zeatin.   A culture medium for tomato cell line culture comprising zeatin and at least one compound of auxin, cytokinin, and saccharide.   The medium for tomato cell line culture according to claim 7 or 8, further comprising α-naphthalene acetic acid, 2,4-dichlorophenoxyacetic acid, and sucrose.