JP3414419B2 - Plant growth / regeneration carrier and plant growth / regeneration method using the same - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a polymeric compound carrier suitable for growing or regenerating a plant, and more specifically, it can be easily recovered or passed over without damaging the grown or regenerated plant. The present invention relates to a carrier for growing or regenerating a plant that can be substituted, and a method for growing or regenerating a plant using the carrier.

[0002]

2. Description of the Related Art In recent years, much attention has been paid to the development of a technique for growing or regenerating a plant having a desired trait.

In recent years, protoplasts have been isolated from plant bodies such as leaves, stems, roots, petals, anthers (pollens) and seedlings by directly growing them.
A variety of techniques have been developed, such as a technique for culturing the protoplasts to regenerate callus or hairy roots for growth. In particular, techniques such as gene transfer or cell fusion are applied at the protoplast stage to cultivate or regenerate more suitable plants. Furthermore, a new breeding method has been developed in which a foreign gene is directly introduced into plant tissues such as leaves, stems, roots, petals and anthers by a gene gun or the like.

[0004] The most important point common to these new breeding methods is that the plants that have acquired the desired trait are efficient,
It is to establish a culture method capable of growing or regenerating.

Cultivation of plants is roughly divided into liquid culture and solid culture using gel such as agar. Liquid culture is a method suitable for large-scale culture because it cultivates plants in suspension.At the same time, subculturing is easily possible, and it is a bioreactor for the production of secretions of plants. Although it has advantages such as being suitable, liquid culture may be difficult depending on the type of plant.

On the other hand, the solid culture method using agar or the like is a method applicable to a wide range of plant species and is particularly suitable for mutant selection, but it has the following major problems. Have a point.

1) It is very difficult to collect or passage a plant grown or regenerated on an agar medium in an agar-free state. 2) When culturing in an agar medium, the nutrient supplementation is performed by diffusion from the outside of the gel, so the efficiency is very poor. 3) The waste products accumulated in the agar medium are also removed by diffusion, which is very inefficient. In particular, it is almost impossible to remove growth inhibitory substances such as polyphenols produced by plants. 4) It is not suitable as a carrier for a bioreactor for the purpose of producing a substance produced by a plant.

The above-mentioned problems are caused by the physical properties of gels such as agar used in conventional solid culture. That is, agar or agarose has a sol-gel transition temperature, exhibits a dissolved state or a sol state at a temperature higher than the transition temperature, and has a property of changing to a gel state at a temperature lower than the transition temperature. Utilizing this property,
Solid culture has been carried out by immersing a plant in an agar or the like in a sol state at a temperature higher than the sol-gel transition temperature, and changing it to a gel state by lowering the temperature.

However, the melting temperature of agar gel is very high, around 90 ° C. (Yamauchi Aizo et al., Polymer O)
Due to ne Point "Functional gel" P29, Kyoritsu Shuppan), the plant is thermally damaged when it is immersed in the agar in a dissolved state. Further, when recovering or subculturing the grown or regenerated plant from the agar medium, it is necessary to raise the temperature to a temperature higher than the sol-gel transition temperature to change the agar to a sol state. Exposed to very large thermal damage.

Therefore, in the conventional solid culture, it was extremely difficult to recover or subculture in an agar-free state due to temperature change. On the other hand, mechanically removing the agar adhering to the plant not only requires a lot of labor, but also may damage the tissues of the fragile plant (especially callus and hairy root). was there.

Since the removal of agar after growth and regeneration has the above-mentioned problems, in the conventional solid culture method, the transplantation of the grown or regenerated plant into the soil actually causes the agar to be removed from the plant. It has been implemented with it attached. However, in this case, since the agar medium contains a lot of nutrients, bacteria,
It has been pointed out that molds and the like are prone to breeding and rot the plants.

On the other hand, in a bioreactor intended to produce a secretion product of a plant, recovery of the secretion product from the gel-like agar medium is much more difficult than the liquid culture method. That is, the high temperature heating during the dissolution of the agar gel containing the secretory substance causes a significant damage to the secretory substance.

In order to alleviate the above problems of the agar gel, sodium alginate has been used as a carrier for growing or regenerating plants. That is, it is a method of immersing a plant in an aqueous solution of sodium alginate and then bringing the solution into contact with a concentrated calcium chloride solution to change the sodium alginate into a gel state.

Although calcium alginate has the advantage of being less toxic, the above method has the following problems.

1) It is necessary to dissolve the calcium alginate gel at the time of collecting or subculturing the plant grown or regenerated in the calcium alginate gel, and EDTA (ethylenediamine tetraacetic acid) used at that time, etc. Calcium chelators are cells,
Damages tissues etc. 2) Calcium alginate gel has low mechanical strength. 3) In a medium having a high phosphoric acid concentration, calcium in a calcium alginate gel reacts with phosphoric acid to form a precipitate of calcium phosphate, which makes culture difficult and at the same time weakens the gel. 4) Since gelation of sodium alginate occurs due to an ion exchange reaction with calcium, the gelation proceeds from the surface of the gel to the inside, and the plants present inside the gel inhibit the uniform gelation reaction, resulting A gel having a uniform structure is easily generated.

As described above, the gels that have been conventionally used for growing or regenerating plant bodies still have unsolved major problems.

[0017]

SUMMARY OF THE INVENTION An object of the present invention is to provide a carrier for growing or regenerating a plant which can easily collect or pass the grown or regenerated plant, and a plant which uses the carrier. Alternatively, it is to provide a reproducing method.

[0018] Another object of the present invention is to provide a material suitable as a carrier for a bioreactor for producing a substance produced by a plant.

[0019]

As a result of earnest research, the present inventor has found that the property is completely opposite to that of the conventional agar, that is,
The use of a specific polymer that reversibly exhibits a sol state (liquid state) at a temperature lower than the sol-gel transition temperature as a carrier for growing and / or regenerating a plant is extremely effective in solving the above-mentioned problems. I found that.

The carrier for growing or regenerating a plant of the present invention is based on the above findings, and more specifically, a polymer having a hydrophobic portion and a hydrophilic portion in the molecule and having a sol-gel transition temperature. It is characterized by containing a compound and reversibly exhibiting a liquid state (sol state) at a temperature lower than the sol-gel transition temperature.

According to the present invention, further, a polymer compound having a hydrophobic portion and a hydrophilic portion in the molecule and having a sol-gel transition temperature is contained, and the liquid state is reversible at a temperature lower than the sol-gel transition temperature. A carrier showing (sol state)
The carrier is mixed with a predetermined medium at a temperature lower than the sol-gel transition temperature, the plant is mixed in the mixture, and the mixture is put in a gel state at a temperature higher than the sol-gel transition temperature to grow or regenerate the plant. A method for growing or regenerating a plant is provided.

In the above-mentioned present invention, the hydrophilic portion of the polymer compound is necessary for the polymer compound to become water-soluble at a temperature lower than the above-mentioned sol-gel transition temperature, and the hydrophobic portion is the sol-gel. It is necessary to change to a gel state at a temperature higher than the transition temperature. In other words, bonds between the hydrophobic moieties are necessary for the formation of gel crosslinks.

The carrier of the present invention utilizes the bond between the hydrophobic moieties in the polymer compound, that is, the property of the hydrophobic bond described below.

Since the hydrophobic bond has a property that its binding force becomes stronger as the temperature rises, the strength of the crosslink and the crosslink density increase as the temperature rises. In the present invention, plant growth or regeneration is carried out. It is possible to change to a gelled state at the temperature of time (temperature higher than the sol-gel transition temperature). Further, in the present invention, the sol-gel transition reversibly occurs due to the property that the temperature dependence of the hydrophobic binding force is reversible.

On the other hand, as described above, the hydrophilic portion in the polymer compound becomes water-soluble at a temperature lower than the temperature at which the polymer compound is grown or regenerated (lower than the sol-gel transition temperature). In order to form a water-containing gel state while preventing the polymer compound from coagulating and precipitating due to excessive increase in hydrophobic binding force at a temperature higher than the above temperature while being necessary for the change. Is also necessary.

The present invention will be described in more detail below.

(Sol-gel transition temperature) In the present invention,
The "sol state", "gel state" and "sol-gel transition temperature" are defined as follows. For the definition, see the literature (Polymer Journal, 18 (5), 411-416.
(1986)).

1 mL of a polymer solution (a polymer solution to be used as a carrier) is placed in a test tube having an inner diameter of 1 cm and kept in a water bath at a predetermined temperature (constant temperature) for 12 hours. After that, when the test tube is turned upside down and the solution / air interface (meniscus) is deformed by its own weight (including the case where the solution flows out), the polymer solution is Defined as "sol state". On the other hand, when the solution / air interface (meniscus) is not deformed by the weight of the solution even when the test tube is turned upside down, the solution is defined as "gel state" at the predetermined temperature. To do.

On the other hand, when the temperature at which the "sol state" transitions to the "gel state" is determined by gradually increasing the above "predetermined temperature" (for example, in steps of 1 ° C), the transition temperature determined by this is calculated. It is defined as the "sol-gel transition temperature" (at this time, the "predetermined temperature" may be lowered by 1 ° C to obtain the temperature at which the "gel state" transitions to the "sol state").

In the carrier of the present invention, the sol-gel transition temperature is higher than 0 ° C. and preferably 60 ° C. or lower, more preferably 4 ° C. or higher and 50 ° C. or lower (particularly 4 ° C. or higher and 40 ° C.).
The following is preferable from the viewpoint of preventing thermal damage to plants. The polymer compound having such a suitable sol-gel transition temperature can be easily selected from the specific compounds described below according to the above-mentioned screening method (sol-gel transition temperature measuring method).

In the method for growing or regenerating a plant of the present invention, the sol-gel transition temperature (a ° C.) described above is the temperature at the time of growing or regenerating the plant (b ° C.), and the grown or regenerated plant is recovered. Alternatively, the temperature (c
It is necessary to set the temperature between (° C). That is, b> a between the three temperatures a ° C., b ° C., and c ° C.
It is necessary to have a relation of> c. More specifically,
(Ba) is preferably 1 to 40 ° C, more preferably 2 to 30 ° C, and (ac) is 1 to 40 ° C, more preferably 2 to 30 ° C.
It is preferably 30 ° C.

(Polymer Compound) The suitable sol-gel transition temperature as described above is, for example, the composition of the hydrophobic portion and the hydrophilic portion in the polymer compound used in the carrier of the present invention, and the hydrophobicity and hydrophilicity. , Molecular weight, etc., respectively.

Specific examples of the polymer compound containing such a hydrophobic portion and a hydrophilic portion include, for example, polyalkylene oxide block copolymers represented by block copolymers of polypropylene oxide and polyethylene oxide; Etherified cellulose such as methyl cellulose and hydroxypropyl cellulose; chitosan derivative (KRHolme, et al, Macromolecules, 24 , 382)
8, (1991)); Pullulan derivative (Degashira, Polyme
r Preprints, Japan, 19 , 936, (1990)) and other modified polysaccharides; poly (N-substituted (meth) acrylamide derivatives, polyvinyl alcohol partial acetic acid, polyvinyl methyl ether, and other temperature-sensitive polymers and water-soluble polymers. Conjugates with hydrophilic polymer compounds (eg Takehisa Matsuda et al., Poly
mer Preprints, Japan 39 (8), 2559, (199
0)) and the like, but not limited thereto.

The concentration of the above-mentioned polymer compound in the present invention varies depending on the sol-gel transition temperature to be set and the like, but when used in a mixture with a predetermined medium as described later, the concentration is generally 0.1 to 30 wt%. , Further 1-20
A concentration of wt% is preferable.

(Medium) In the present invention, a known medium (for growing and / or regenerating plants) which is substantially free of agar is used as a medium or a culture medium to be used in combination with the above-described polymer compound. The culture solution can be used without particular limitation.

(Plant growth / regeneration method) A preferred example of a method for growing or reproducing a plant using the above-described polymer compound is described below.

First, the above-described polymer compound is uniformly dissolved in a desired medium at a temperature lower than the sol-gel transition temperature of the polymer compound. Next, a plant solution comprising leaves, stems, roots, petals, petals, anthers (pollens), seedlings, etc., or callus, hairy roots or protoplasts regenerated from the plant, in a medium solution of the polymer compound. A plant consisting of Next, by setting the temperature of the plant-dispersed solution to a temperature higher than the sol-gel transition temperature, a gel-like body having a desired shape can be produced, and the plant can be grown or regenerated.

On the other hand, the recovery or passage of the plant grown or regenerated in the gel can be easily carried out by lowering the temperature to a temperature lower than the sol-gel transition temperature and re-dissolving the gel.

Further, supplementation of nutrients deficient in the gel medium, or removal of waste products that interfere with growth or regeneration also dissolves the old gel due to the above-mentioned temperature decrease,
After removal, the plant may be transplanted to a new medium. In the present invention, it is possible to easily continue the gel culture in this way.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is determined by the description of the scope of claims and is not limited by the following examples. Absent.

[0041]

Example 1 Pluronic F-127 (manufactured by Asahi Denka Kogyo Co., Ltd.), which is a block copolymer of polypropylene oxide and polyethylene oxide, was cooled with ice at a concentration of 20 wt%.
Tobacco cell culture medium containing no agar (Hirai Atsushi et al.
Biochemistry Experimental Method 16, "Introduction to Plant Cell Breeding" P15,
(1969), Academic Publishing Center). The sol-gel transition temperature of this solution was 21 ° C as determined by the method described above. The solution was then autoclaved.

Tobacco (Nicotina glut) grown for 4 to 5 months
Inosa) stems are cut into 4 to 5 cm, washed with detergent and tap water, then dipped in 70% ethanol aqueous solution for 30 seconds and 1%
After sterilizing by immersing in an aqueous solution of sodium hypochlorite for 7 minutes, it was washed several times with sterile distilled water.

Next, the stem was cut into pieces of about 5 mm with a sterilized razor, and a cork borer having a diameter of about 3 mm was inserted into the section to collect the marrow tissue, and the marrow tissue was further cut into about 2 to 3 mm. All of the above operations were performed aseptically.

Next, several marrow tissue sections obtained by the above method were uniformly dispersed in about 10 mL of ice-cooled tobacco culture medium containing Pluronic F-127,
The dispersion was poured into a petri dish having a diameter of 6 cm and the temperature was adjusted to about 2
The solution was gelled by raising to 8 ° C. and the marrow tissue was embedded in the gel. Then 100 at about 28 ° C
When cultured for 15 days under artificial light irradiation of 0 to 3000 lux, good callus formation was observed from each marrow tissue section.

Next, in order to recover the callus formed in the gel, the gel was dissolved by cooling the gel under ice cooling for about 3 minutes, and then the callus was isolated by gentle centrifugation. did. By further adding 0.5 M mannitol solution and resuspending the mixture, centrifugation was performed to recover the callus of tobacco from which Pluronic F-127 was completely removed.

Example 2 10 g of Pluronic F-127 used in Example 1 was dissolved in 30 mL of dry chloroform and coexisted with phosphorus pentoxide.
Hexamethylene diisocyanate (0.13 g) was added, and the mixture was reacted under reflux of boiling point for 6 hours. After removing the solvent under reduced pressure, the residue was dissolved in distilled water and subjected to ultrafiltration dialysis using an ultrafiltration membrane having a molecular weight cutoff of about 100,000 to recover an aqueous solution containing only the high molecular weight polymer. The obtained aqueous solution was freeze-dried to obtain a polymer of Pluronic F-127. The polymer was dissolved in distilled water at a concentration of 10 wt% under ice cooling.

When this aqueous solution is gradually heated,
The viscosity gradually increased from 21 ° C., completely solidified at about 27 ° C., and changed into a gel state. When the gel cools, about 21
It turned into an aqueous solution at ℃. This change was repeatedly observed reversibly.

The Pluronic F-127 polymer was dissolved in the tobacco medium used in Example 1 at a concentration of 10 wt% under ice cooling and sterilized by autoclaving.

2 to 3 produced in exactly the same manner as in Example 1
A few 3 mm-sized tobacco pulp tissues were dispersed in the above-mentioned Pluronic F-127 polymer tobacco medium solution under ice cooling, and then cultured for 7 days in the same manner as in Example 1. Good callus formation was observed. The callus could be easily recovered by the same method as in Example 1.

Example 3 Protoplasts were isolated and purified from the tobacco callus obtained in Example 2 by the following method.

Add 80 mL of 0.5 M mannitol solution to the Cell.
ulase Onozuka (Yakult Pharmaceutical Industry) 1g, Macerozyme
(Yakult Pharmaceutical Industry) 200 mg, Pectolyase 10 mg
After each was dissolved, 0.5 M mannitol solution was added to bring the total volume to 100 mL, centrifugation was performed at 5,000 g for 10 minutes, and the supernatant was sterilized by filtration to prepare an enzyme solution for protoplast isolation.

Next, 10 mL of the enzyme solution was placed in a petri dish having a diameter of 6 cm, 500 mg of the collected callus obtained in Example 2 was added, the petri dish was sealed with Parafilm (American CAN), and shaken at 40-50 rpm. 3 down
Incubated for hours. After confirming that protoplasts were isolated using an inverted microscope, the content liquid of the petri dish was filtered through a funnel made of mesh (Becton Dickinson) to remove cell debris. Then, the suspension of protoplasts was spun down at 150 g for 2 minutes to obtain protoplasts as a precipitate. 0.5 for protoplast precipitation
M mannitol solution was added, resuspended, and centrifuged. The protoplast was washed by repeating this operation twice.

Then, the agar-free medium for tobacco protoplasts containing 10 wt% of Pluronic F-127 polymer in the above-mentioned tobacco protoplasts (Atsushi Hirai et al., Biochemistry Experimental Method 16, "Introduction to Plant Cell Breeding", P41,
(1969), Academic Publishing Center), under ice cooling, about 10
Dispersed at a concentration of ⁶ / mL. 4 mL of the protoplast suspension was poured into a petri dish having a diameter of 6 cm, and the temperature was raised to 28 ° C to form a gel in which protoplasts were embedded.

When the gel was cultured for 5 days at 28 ° C. under artificial light irradiation of about 500 lux, colonies, that is, callus was formed in the gel. Then, the regenerated callus could be easily recovered from the gel in the same manner as in Example 1. Regenerated callus with 0.1% Calcofluor Whi
When stained with te ST (manufactured by American Cyanamide) and observed with a fluorescence microscope, it was confirmed that the cell wall was regenerated in cells forming callus.

[0055]

As described above, according to the present invention, the embedding of a plant in a gel and the recovery of the plant from the gel can be easily performed by, for example, a temperature change within the physiological temperature range of the plant. In addition, it can be carried out without damaging the plant body at all. Therefore, according to the present invention, the problems of the conventional gel culture method using agar or the like can be completely solved.

Claims (4)

(57) [Claims] 1. A polymer compound having a hydrophobic portion and a hydrophilic portion in the molecule and having a sol-gel transition temperature, and reversibly in a liquid state (sol state) at a temperature lower than the sol-gel transition temperature. And a carrier for growing or regenerating a plant. 2. The carrier for growing or regenerating a plant according to claim 1, wherein the sol-gel transition temperature is higher than 0 ° C. and 60 ° C. or lower. 3. A polymer compound having a hydrophobic portion and a hydrophilic portion in the molecule and having a sol-gel transition temperature, and reversibly showing a liquid state (sol state) at a temperature lower than the sol-gel transition temperature. The carrier is mixed with a predetermined medium at a temperature lower than the sol-gel transition temperature of the carrier, the plant is mixed into the mixture, and the mixture is put in a gel state at a temperature higher than the sol-gel transition temperature to grow the plant. Alternatively, a method for growing or regenerating a plant to be regenerated. 4. The method according to claim 3, wherein the gel containing the grown or regenerated plant is put into a liquid state (sol state) at a temperature lower than the sol-gel transition temperature, and the plant is collected and / or passaged. Method for raising or regenerating plant body of.