JPH11196863A - Prevention of browning of plant cultured cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent the browning of a plant cell, the deterioration of a cell proliferation rate, or the like on the culture of the cell of a woody plant such as a needle-leaved tree belonging to the order Taxales, Taxodiales, Pinaceae or the like by adding a specific phytosulfokine to a culture medium. SOLUTION: This method for preventing the browning of a plant cell comprises adding a phytosulfokine of formula I [R<1> and R<2> are each independently SO3 H or H; X is an α-amino acid or a single bond; Y is OH or NH2 ; Z<1> is an α-amino acid; Z<2> is an α-amino acid or a single bond], preferably a phytosulfokine-α of formula II preferably in a concentration of 10<-9> to 10<-4> M. The phytosulfokine is isolated from a culture filtrate of asparagus cells or the like, or is prepared, for example, by a method for synthesizing a peptide and subsequently introducing a sulfonyl group and the like to the peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to a method for suppressing the formation of browning substances in woody plant cells in cell culture.

[0001]

2. Description of the Related Art Practical application of plant cell culture to breeding and production of useful secondary metabolites has been studied, but there have been very few cases of industrial success. One of the causes is, for some plants, particularly woody plants, a reduction or cessation of cell growth due to the generation of browning substances and a decrease in productivity of desired secondary metabolites.

[0002] This browning substance (which may have a red or reddish brown color depending on the plant species) is often a polyphenolic compound or a polymer thereof, and the stress or irritation exerted on the cell limits the permissible limit of the cell. It is thought that by exceeding this, the secondary metabolism of the phenylpropanoid system is activated to form. The aforementioned defects related to cell growth and production of secondary metabolites are due to the inhibition of various enzyme activities in the cells by browning substances accumulated in the medium, or in extreme cases, the inactivation of the enzymes themselves. Yes, as for the production of secondary metabolites, the direction of biosynthesis of secondary metabolites is fixed to the phenylpropanoid system, and the production of secondary metabolites in other biosynthetic pathways is suppressed It is thought that.

[0003] Many studies have been made on the prevention of browning of plant cells, and it has been reported that the addition of an antioxidant such as ascorbic acid or cytokinins is effective. However, these effects are in many cases species-specific and at present they do not solve the problem 100%.

[0004] Stress and irritation on cells are likely to appear immediately after transplantation of cells into fresh medium, which means that various environments surrounding the cells, such as the osmotic pressure of the medium, nutrient and phytohormone concentrations, dissolved oxygen, This indicates that such a rapid change can act as these stresses and stimuli. Based on this idea, in order to minimize the effects of these stresses and stimuli, conditioning is a process in which cells are transplanted to a fresh medium at a certain rate by mixing the old medium that had been used for culturing the cells. Is taken. It is known that conditioning can relieve these stresses and stimuli to a certain extent, thereby reducing the induction period of cell proliferation and providing other benefits (Stuart, R. & Street, HE, J. Exp. Bot. 20, 556
-571 (1969)). It is also known that physical factors such as stirring and temperature during culture can also act as stress and stimuli. Factors of the effectiveness of conditioning include activation of cell proliferation,
It has long been thought to contain substances that directly trigger cell division, but the substance has long been unknown.

On the other hand, Sakagami and Matsubayashi of the Faculty of Agriculture of Nagoya University isolated a peptidic cell growth factor from a medium for conditioning asparagus cells and named it phytosulfokine. Has been shown to promote cell growth in monocotyledonous plants, including growing lilies and gramineous plants (Matsubayashi
Y. et al, Proc. Natl. Acad. Sci. USA 93, 7623-7627
(1996), MatsubayashiY. Et al, Proc. Natl. Acad. S
ci. USA 94, 13357-13362 (1997)). Patents for the compounds have also been filed (WO 96/32409 and WO 96/32409).
96/32410).

The phytosulfokines isolated and identified so far include phytosulfokine-α, phytosulfokine-β, and phytosulfokine-
α [1-3] and the like, each of which is represented by the following general formula I:

Embedded image [In the formula, R ¹ and R ² each independently represent a SO ₃ H or H; X represents alpha-amino acid or a single bond; Y represents a hydroxyl group or an amino group; Z ¹ is alpha- Represents an amino acid; Z ² represents an α-amino acid or a single bond].

A phytosulfokine compound can be isolated from a culture filtrate of asparagus cells or the like, or a method of synthesizing a peptide and introducing a sulfonyl group or the like to the peptide using an enzyme or the like (Matsubayashi Y. et al,
Proc. Natl. Acad. Sci. USA 93, 7623-7627 (199
6)), a method of chemically synthesizing from amino acids such as sulfated tyrosine (Matsubayashi Y. et al, Biochem. Biophys. R
es. Commun. 225, 209-214 (1996)). Methods for preparing these phytosulfokine compounds are disclosed in detail in the above-mentioned articles and patents.

[0008] It is known that the growth rate of plant cells decreases when the density per culture medium falls below a certain level, or it becomes impossible to grow in extreme cases. The specification states that the addition of phytosulfokines to the medium can significantly reduce the density limit required for such plant cell growth. However, no specific effect on woody plant cells was known, and it was unexpected that phytosulfokine was effective in preventing browning.

[0009]

Means for Solving the Problems The present invention provides a method for culturing woody plant cells of the following general formula I:

Embedded image [In the formula, R ¹ and R ² each independently represent a SO ₃ H or H; X represents alpha-amino acid or a single bond; Y represents a hydroxyl group or an amino group; Z ¹ is alpha- It represents an amino acid; Z ^2, by adding a compound represented by] representing the α- amino acid or a single bond to the medium, a method of preventing the browning of the plant cell.

The phytosulfokines used in the present invention include the following general formula I:

Embedded image [In the formula, R ¹ and R ² each independently represent a SO ₃ H or H; X represents alpha-amino acid or a single bond; Y represents a hydroxyl group or an amino group; Z ¹ is alpha- Represents an amino acid; Z ² represents an α-amino acid or a single bond], among which the following general formula II:

Embedded image [Wherein X represents an α-amino acid or a single bond;
Which represents a hydroxyl group or an amino group], or the following general formula III:

Embedded image [Wherein, Y represents a hydroxyl group or an amino group; Z ² represents α
-Representing an amino acid or a single bond].

In the above general formulas I to III, the α-amino acid represented by X is exemplified by glutamine (Gln);
^{As the} α-amino acid represented by ¹ , isoleucine (Il
e) is exemplified, and as an α-amino acid represented by Z ² ,
Threonine (Thr), serine (Ser) and the like can be exemplified.

As specific examples of the compounds represented by the above general formulas I to III, in the general formula I, R ¹ and R ² are SO ₃ H, X is glutamine (Gln), and Y is OH Phytosulfokine-α (Compound A, SEQ ID NO: 1) wherein Z ¹ is isoleucine (Ile) and Z ² is threonine (Thr);

Embedded image

In the general formula I, R ¹ and R ² represent SO ₃
Phytosulfokine-β wherein H is X is a single bond, Y is OH, Z ¹ is isoleucine (Ile), and Z ² is threonine (Thr) (Compound B, SEQ ID NO: 2) ;

Embedded image

In the general formula I, R ¹ and R ² represent SO ₃
A phytosulfokine-α [1-3] wherein H is X, a single bond, Y is OH, Z ¹ is isoleucine (Ile), and Z ² is a single bond (compound C);

Embedded image Phytosulfokine-α (compound A) is particularly preferred.

In the present invention, woody plants can be used as a plant in which the above-mentioned phytosulfokine can be used for browning prevention.
A large amount of xylem is formed in stems and roots by hypertrophic growth, and the cell wall is one that becomes lignified and becomes strong,
It is not specified taxonomically.

In the present invention, the phytosulfokines that can be used for browning prevention include coniferous (coniferous) plants including yew, cedar and pine. Among them, a Yew genus plant of the Taxus family Yew is particularly preferred.

As a yew plant, the yew tree (T
axus baccata LINN), yew (T. cuspidata SIEB. et Z
UCC.), T. cuspidata SIEB. Et ZUCC.var
nana REHDER), T. brevifolia NUT
T), Canada yew (T. canadensis MARSH), Chinese yew (T. chinensis), Himalayan yew (T. wallichian)
a), T. media, etc., of which Yew yew and Taxus media are preferred.

In the present invention, the phytosulfokine can be used in either a liquid medium or a solid medium,
The addition concentration is 10 ⁻⁹ to 10 ⁻⁴ M, preferably 10 ⁻⁹ M.
^-8 to 10 ^-5 M can be exemplified.

When cells of these plants are cultured in the presence of a phytosulfokine compound, cell growth can be directly promoted as conventionally known for monocotyledonous plants. Alternatively, by suppressing the production of browning substances, the inhibition of cell growth can be released. This suppression release is based on a mechanism completely different from the cell growth promoting effect conventionally known for monocotyledonous plants, and can be said to be a phenomenon unique to woody plants.

In particular, when a plant cell is subjected to mutagen treatment, introduction of a foreign gene, or selection of a resistant strain by treatment with a drug such as an antibiotic, the cell is exposed to strong stress, and the generation of a browning substance occurs. In such a case, the addition of phytosulfokine to the medium is advantageous in that the growth of cells increases directly and indirectly, and the probability of obtaining desired cells increases. is there.

Similarly, in the presence of a phytosulfokine compound, the direction of secondary metabolism is not fixed to phenylpropanoid, so that a desired secondary metabolite can be efficiently produced. In the method according to the present invention, as a secondary metabolite that can be expected to be efficiently produced,
Examples include taxane diterpenes such as taxol produced by Taxus plants, tropolone derivatives such as hinokitiol produced by Hinoki and Hiba, ginkgolides and bilobalides produced by Ginkgo biloba.

Preferred examples of the present invention for preventing browning of cells with phytosulfokines include the following methods. A part of the plant, for example, roots, growing points, leaves, stems, after sterilization of plant pieces collected from seeds and the like, placed on a solid medium such as Murashige-Skoog medium hardened with gellan gum, 10 ~ 35 ℃ After about 14 to 60 days, callus is generated from a part of the tissue piece. When the culture thus obtained is subcultured, the growth rate gradually increases and a stabilized culture can be obtained. Here, the term "stabilized culture" refers to a culture having a property of maintaining a state in which undesired organ differentiation or callus does not occur during culture and having a uniform growth rate of the culture.

The stabilized culture is transferred to a liquid medium suitable for growth, for example, Murashige Skoog's liquid medium, and grown. The growth rate is further increased in the liquid medium. The temperature for the culture of the present invention is usually about 10 to
About 35 ° C, especially about 23-28 ° C, is preferred because of the high growth rate. The culture period is preferably 7 to 42 days. Cells cultured in the presence of phytosulfokine in this manner are regenerated into plants by a known method,
It can be used for breeding applications such as selection of excellent lines.
In addition, secondary metabolites of interest can be separated from cultured cells and / or culture medium by a method such as extraction with an organic solvent.

[0024]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, Comparative Examples and Reference Examples, but the scope of the present invention is not limited to these Examples. In addition, the presence or absence of generation of a browning substance was visually determined.

Example 1 A solid medium (0.25% by weight of gellan gum) of Woody Plant Medium to which naphthalene acetic acid was added to a concentration of 10 ^-5 M was prepared in advance.
A portion of the stem of Taxus media (Taxus media LINN) sterilized with a 2% antiformin solution or a 70% ethanol solution was placed on a bed and allowed to stand still at 25 ° C. in a dark place to obtain a callus. Next, 1 g of this callus (fresh weight) was transferred to an Erlenmeyer flask containing 20 ml of a liquid medium of Woody Plant Medium to which the above-mentioned components were added at the same concentration, and subjected to vortex culture (amplitude: 25 mm, 100 rpm) on a rotary shaker.
Planting was continued every day to increase the growth rate of the callus.

The cultured cells thus obtained were fractionated using a stainless mesh to obtain a Φ100-180 μm fraction containing a large amount of cell mass consisting of about 5 to 10 cells. The cells of the obtained fraction were suspended in Woody Plant Medium. The cell suspension was adjusted to 10 ⁻⁶ M ^{phytosulfokine-} so that the final concentration was 5 × 10 ⁴ cells / ml.
Suspend in Woody Plant Medium containing α and 0.4% low melting point agarose, cool in a sterile plastic Petri dish (Φ60 x 15 mm), and cure at 25 ° C in a dark place.
The culture was allowed to stand for a day. The results are shown in Table 1 and FIG.

[Comparative Example 1] The procedure of Example 1 was repeated, except that the culture was carried out in a medium to which phytosulfokine-α was not added at the start of the culture. The result is displayed
1 and FIG.

Example 2 In Example 1, the concentration of phytosulfokine-α was 10 ^{−10 to} 3.3 × 10 ⁻⁶ M.
The operation was performed in the same manner as in the example, except that The result is shown in FIG. In addition, the effect of suppressing browning appeared at a concentration of 1.0 × 10 ⁻⁹ M or more.

Example 3 The procedure of Example 1 was repeated except that the period of the stationary culture was changed to 7, 12, 14, and 17 days. The result is shown in FIG. Note that no browning substance was generated.

Comparative Example 2 The procedure of Example 3 was repeated, except that phytosulfokine-α was not added. The result is shown in FIG. Static culture period 12
After the day, browning of the cells was observed, and the degree of browning became remarkable as the number of days passed.

Example 4 In Example 1, the cell suspension contained 10 ^-6 M phytosulfokine-α, and contained 0.4%
The same operation as in this example was performed, except that the culture was performed by swirling at a rate of 80 rpm / min in a liquid medium of Woody Plant Medium containing no low melting point agarose. The results are shown in Table 2 and FIG. Further, the culture after 2 months of culture is centrifuged into cells and a liquid medium, and 500 nm derived from a brown substance is centrifuged.
The spectrum of the medium component was measured using the near absorption as an index. The result is shown in FIG.

Comparative Example 3 The procedure of Example 4 was repeated except that phytosulfokine-α was not added. The results are shown in Table 2, FIG. 3 and FIG.

[0033]

[Table 1]

[0034]

[Table 2]

[0035]

According to the present invention, browning of cells can be prevented in cell culture of woody plants.

[0036]

[Sequence list] SEQ ID NO: 1 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Sequence type: Peptide Sequence features: Symbol indicating feature: Modified-site Location: 1 Determine feature Method performed: E Other information: Xaa represents O-sulfotyrosine. Symbol representing characteristics: Modified-site Location: 3 Method for determining characteristics: E Other information: Xaa represents O-sulfotyrosine.

SEQ ID NO: 2 Sequence length: 4 Sequence type: number of amino acid chains: single-chain Sequence type: peptide Sequence characteristics: Symbol indicating characteristics: Modified-site Location: 1 Characteristic determined Method: E Other information: Xaa represents O-sulfotyrosine. Symbol representing characteristics: Modified-site Location: 3 Method for determining characteristics: E Other information: Xaa represents O-sulfotyrosine.

[0038]

[Brief description of the drawings]

FIG. 1. PSK-α (Phytolsulfokine-
1 is a graph showing the cell division promoting activity of α).

FIG. 2. PSK-α (Phytolsulfokine-
(a) is a graph comparing the time-dependent changes in cell proliferation when treated (Example 3, black circles) and untreated (Comparative Example 2, open circles).

FIG. 3 shows absorption spectra of medium components of Example 4 and Comparative Example 3.

FIG. 4 is a photograph of the cultured cells obtained in Example 1 and Comparative Example 1.

FIG. 5 is a photograph of the cultured cells obtained in Example 4 and Comparative Example 3.

Claims (8)

[Claims] 1. A method for culturing woody plant cells, which comprises the following general formula I: [In the formula, R ¹ and R ² each independently represent a SO ₃ H or H; X represents alpha-amino acid or a single bond; Y represents a hydroxyl group or an amino group; Z ¹ is alpha- Wherein Z ² represents an α-amino acid or a single bond] to the medium. 2. The compound to be added to the medium has the following general formula:
II: [Wherein X represents an α-amino acid or a single bond;
Represents a hydroxyl group or an amino group]. 3. The method according to claim 2, wherein the compound added to the medium is phytosulfokine-α. 4. The compound to be added to the medium has the following general formula:
III: [Wherein, Y represents a hydroxyl group or an amino group; Z ² represents α
-Represents an amino acid or a single bond]. 5. The method according to claim 1, wherein the target plant is a coniferous plant. 6. The method according to claim 5, wherein the target plant is a Taxus plant. 7. The target plant is a yew tree (Taxus).
7. The method according to claim 6, which is baccata) or T. media. 8. The method according to claim 1, wherein the concentration of the compound represented by the formula (I) is 10 ^-9 to 10 ^-4 M.