JPH0856654A - Stabilization of protoplast and artificial cell wall-coated protoplast - Google Patents

Abstract

PURPOSE: To stabilize protoplast useful in the cell technology and biotechnology, etc., by culturing protoplast in a hydrophobicized polysaccharide-contg. medium to coat its cell membrane with the hydrophobicized polysaccharide. CONSTITUTION: The protoplast of vegetable cells (pref. carrot protoplast) is cultured in a medium containing a hydrophobicized polysaccharide (pref. partially hydrophobicized pullulan, amylose, mannan, amylopectin, dextran or xylogl-ucan phosphate) to coat its cell membrane with the polysaccharide to temporarily form artificial cell wall, thus stabilizing the protoplast.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for stabilizing protoplasts. More specifically, the present invention relates to a new method for stabilizing protoplasts, which is widely useful in fields such as cell engineering and biotechnology, and an artificial cell wall-coated protoplast stabilized by this method.

[0002]

[Prior art and its problems] Protoplast
t) is a protoplast obtained by removing the cell walls of bacteria, fungi, plant cells, etc. by enzymatic treatment, etc., and maintains various cellular activities under appropriate osmotic pressure conditions while introducing polymeric substances and particles. Alternatively, it has an excellent property that operations such as cell fusion can be performed.

Therefore, protoplasts are widely used, for example, for producing transformants by introducing exogenous DNA and selecting cells having a specific genetic character. In the case of protoplasts of plant cells, their usefulness is also recognized as a material for solid subdivision for the purpose of mass breeding of excellent plant strains. However, protoplasts
As it is, it has problems such as low cell stability and easy regeneration of cell wall.In actual use, careful preparation and preparation of protoplasts are necessary. It wasn't always easy to use.

The present invention has been made in view of the above circumstances, and provides a method for increasing the stability of protoplasts without affecting the properties of the protoplasts, and a protoplast stabilized by this method. It is intended to be provided.

[0005]

Means for Solving the Problems The present invention, as a solution to the above-mentioned problems, comprises culturing protoplasts in a hydrophobized polysaccharide-containing medium and coating the cell membrane thereof with hydrophobized polysaccharides to stabilize the protoplasts. Method is provided. The present invention also provides an artificial cell wall-coated protoplast whose cell membrane is coated with a hydrophobized polysaccharide.

Further, in the present invention, one of the preferred embodiments is that the hydrophobized polysaccharide is partially hydrophobized pullulan or xyloglucan phosphate, and the protoplasts are plant cell protoplasts. It also provides carrot protoplasts whose cell membranes are partially hydrophobized and coated with pullulan or xyloglucan phosphate.

The present invention will be described in more detail below. The protoplasts targeted by the present invention are protoplasts of bacteria, fungi (yeast, filamentous fungi, etc.) or plant cells, and can be prepared according to known methods. For example, when preparing a protoplast of a plant cell, a cell wall adhesive substance (hemicellulose, pectin polysaccharide, etc.) is first enzymatically decomposed into single cells, and then the cell wall containing cellulose as a main component is decomposed by an enzymatic treatment such as cellulase. These two steps of enzyme treatment can be performed simultaneously.

[0008] The protoplasts thus isolated and prepared are also cultured by a known method. In the present invention, a hydrophobized polysaccharide is added to the medium. The hydrophobized polysaccharide is, for example, a natural polysaccharide such as pullulan, amylose, mannan, amylopectin, dextran, and xyloglucan phosphate modified with a hydrophobic group such as cholesterol or palmitoyl. , The above-mentioned natural polysaccharide and a hydrophobic group can be self-assembled in an aqueous solution (Macromolec
ules, 25, 5665-5670, 1992; Physicochemical Charact
erization of Cholesterol-Bearing Polysaccharides in
Solution: Organized Solutionsedited by Friberg,
SE and Lindman, B., Marcel Dekker. Inc., 290-30
4,1992). As an example of such a hydrophobized polysaccharide, the chemical formula of pullulan modified with a cholesterol group is shown below.

[0009]

Embedded image

By the method as described above, the cell membrane of protoplasts can be coated with the hydrophobized polysaccharide (see FIG. 1), which makes it possible to manipulate the originally unstable protoplasts in an extremely stable state. For example, normal protoplasts are calcium ionophore A23
It is dissolved by 187 (Plant Science, 69, 135-
138, 1990), the artificial cell wall-coated protoplasts stabilized by the method of the present invention show high resistance to A23187 as shown in the following examples.

Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to the following examples.

[0012]

Example 1 (Preparation of carrot protoplasts and their stability
Reduction) was first prepared protoplasts by the following procedure from carrot cells. The carrot suspension culture cells used as the material were derived from the carrot variety Kuroda-gosan, and contained Murasige and Skoog medium [sucrose 3%, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg / L, kinetin 0.1 mg / L]. L content: p
H was subcultured at 5.8 before autoclaving).
Transplanting is done every two weeks, 120 round trips per minute, amplitude 5
The cells were cultured at 6 cm and 24 ° C. 6 days after subculture, cells were collected by filtration in a clean bench, Murasige and Skoo
g After washing once with a medium (containing 0.25 M mannitol and 0.25 M sorbitol: pH 5.6), it was collected by filtration in a sterilized Erlenmeyer flask. Enzyme solution (Cellulase Onozuka R 10: 1.
A washing solution containing 8% and Driselase: 0.2%) was filled in a syringe, and a sterilizing filter was attached to the tip of the injection needle, and the enzyme solution was added to the Erlenmeyer flask while sterilizing by filtration. After sealing the flask, it was placed in a rotary infiltrator and allowed to infiltrate at 24 ° C. for 18 hours in the dark. The sample dispersion was filtered with a nylon filter having a pore size of 40 μm, and the filtrate was mixed with 50 g of 5
After centrifuging, the supernatant was removed and dispersed with a washing solution, then gently added onto 6% Ficoll sucrose solution (0.5 M) and centrifuged at 200 × g for 5 minutes. Protoplasts agglomerated at the two-phase interface. The supernatant was removed, protoplasts were placed in a centrifuge tube with a capacity of 10 ml, dispersed with a washing solution, and centrifuged at 50 × g for 5 minutes. This operation was repeated 3 times. Finally, a small amount of protoplast culture medium (0.4 M mannitol added to the above cell culture medium) was dispersed to prepare carrot cell protoplasts.

Then, the protoplasts were added to pullulan (CHP) modified with a cholesterol group (CHP) or xyloglucan phosphate (CHX-P) at a rate of 0.3 mg / ml, respectively, in the protoplast culture medium at 24 ° C. Cultured in the dark for ~ 3 hours. Also, as a control, protoplasts were similarly cultured in a culture solution containing no CHP or CHX-P.

The number of each protoplast cultured as described above was measured with a fluorescence microscope by the Fluorescein diacetate method, and the survival rate of each was determined. The results are shown in Table 1.
And the protoplasts cultured in the CHP- or CHX-P-containing medium showed higher survival rate than the control.

[0015]

[Table 1]

Furthermore, the state of the CHP coating is changed to Fluorescein.
CHP modified with isothiocyanate (CHP-FIT
C) Confocal fluorescence microscope (Bio-Rad)
Observed at. That is, CHP-FITC is 0.6 mg /
Protoplasts were cultured in the dark at 24 ° C. for 3 hours in a protoplast culture medium added at a rate of ml. The result is shown in Figure 2.
As shown in the micrograph of FIG.
It was confirmed to be clearly covered by FITC. Example 2 (Stabilization test against A23187) CHP coated protoplasts and C prepared in Example 1
The HX-P coated protoplasts were tested for stability against the calcium ionophore A23187.

The results are as shown in FIGS. 3 and 4. Note that FIG. 3 shows coated protoplasts (CHP, CHX in a medium containing A23187 at a concentration of 20 μM).
-P) and the control (Control) non-solubilization rate is shown with time, and FIG. As is clear from these results, the artificial cell wall-coated protoplasts whose cell membrane was coated with the hydrophobized polysaccharide by the method of the present invention exhibited much higher stability than the untreated control. Example 3 (resistance to intracellular influx of extracellular calcium
(Test) The CHX-P-coated protoplasts prepared in Example 1 were tested for resistance to calcium influx in the presence of A23187.

The results are as shown in FIGS. 5 and 6.
5 shows the non-dissolution rate of each protoplast after culturing for 2 hours in a medium containing A23187 (20 μM) and calcium ions (0 to 10 mM), and FIG. 6 shows A231.
87 (20 μM) and polysaccharides (0.0 to 1.2 / mg / m)
The non-dissolution rate of each protoplast after 2 hours of culture in a medium containing 1) is shown.

As is clear from these results, the artificial cell wall-coated protoplasts of the present invention showed superior resistance to extracellular influx of calcium as compared with the untreated control. Example 4 (Test for resistance to cellulase in culture solution) Regarding the CHP-coated protoplasts prepared in Example,
Resistance to cellulase, a cellulolytic enzyme, was tested.

Carrot protoplasts were cultured in the same protoplast culture solution (containing 0.3 mg / ml of CHP) as in Example 1 for 3 hours at 24 ° C. in the dark, and then cellulase (ONOZUKA R-10) was added at 0.3 mg / ml. It was added to the culture medium at a ratio of ml. After culturing for a certain period of time in this state, F
The survival rate of protoplasts was measured by a fluorescence microscope by the luorescein diacetate method. As a control, the protoplasts cultured in the CHP-free culture medium were also measured for survival rate in the cellulase-containing culture medium.

The results are shown in Table 2, and it was confirmed that the artificial cell wall-coated protoplasts of the present invention have high stability against the degrading enzyme cellulase.

[0022]

[Table 2]

[0023]

As described in detail above, according to the present invention, it becomes possible to stabilize protoplasts which are originally extremely unstable with respect to calcium influx and cell wall degrading enzymes. The application range of protoplasts in the fields of engineering and biotechnology will be greatly expanded.

[Brief description of drawings]

FIG. 1 is a schematic view illustrating the method for stabilizing protoplasts according to the present invention.

FIG. 2 is a fluorescence micrograph showing CHP coating a cell membrane of an artificial cell wall-coated protoplast of the present invention.

FIG. 3: Artificial cell wall coated protoplasts (CHP, CHX) in medium containing A23187 at a concentration of 20 μM.
-P) shows the change over time in the non-dissolution rate of the control (Control).

FIG. 4 shows the non-dissolution rate after 2 hours of culture at each concentration of A23187.

FIG. 5 shows the non-dissolution rate of each protoplast after 2 hours of culture in a medium containing A23187 (20 μM) and calcium ions (0 to 10 mM).

FIG. 6 A23187 (20 μM) and polysaccharides (0.0-
(1.2 / mg / ml) shows the non-dissolution rate of each protoplast after 2 hours of culture in a medium containing (1.2 / mg / ml).

[Procedure amendment]

[Submission date] April 6, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction content]

[Document name] Statement

Title: Stabilization method for protoplasts and artificial cell wall coated protoplasts

[Claims]

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for stabilizing protoplasts. More specifically, the present invention relates to a new method for stabilizing protoplasts, which is widely useful in fields such as cell engineering and biotechnology, and an artificial cell wall-coated protoplast stabilized by this method.

[0002]

[Prior art and its problems] Protoplast
t) is a protoplast obtained by removing the cell walls of bacteria, fungi, plant cells, etc. by enzymatic treatment, etc., and maintains various cellular activities under appropriate osmotic pressure conditions while introducing polymeric substances and particles. Alternatively, it has an excellent property that operations such as cell fusion can be performed.

Therefore, protoplasts are widely used, for example, for producing transformants by introducing exogenous DNA and selecting cells having a specific genetic character. In the case of protoplasts of plant cells, their usefulness is also recognized as a material for solid subdivision for the purpose of mass breeding of excellent plant strains. However, protoplasts
The intact have the problem of cell stability have very low, because it requires great care in the preparation and the culture of the protoplasts in the actual use, be utilized for the purposes as described above necessarily It wasn't easy.

The present invention has been made in view of the above circumstances, and provides a method for increasing the stability of protoplasts without affecting the characteristics of the protoplasts, and a protoplast stabilized by this method. It is intended to be provided.

[0005]

SUMMARY OF THE INVENTION The present invention, as to solve the above problems, the protoplasts were cultured in hydrophobized polysaccharide-containing medium, to coat the cell membrane with a hydrophobic polysaccharide, human
It is temporarily form artificially cell wall to provide a method of stabilizing protoplasts characterized by Rukoto. The present invention also provides an artificial cell wall-coated protoplast whose cell membrane is coated with a hydrophobized polysaccharide.

Further, in the present invention, one of the preferred embodiments is that the hydrophobized polysaccharide is partially hydrophobized pullulan or xyloglucan phosphate, and the protoplasts are plant cell protoplasts. It also provides carrot protoplasts whose cell membranes are partially hydrophobized and coated with pullulan or xyloglucan phosphate.

The present invention will be described in more detail below. The protoplasts targeted by the present invention are protoplasts of bacteria, fungi (yeast, filamentous fungi, etc.) or plant cells, and can be prepared according to known methods. For example, when preparing a protoplast of a plant cell, a cell wall adhesive substance (hemicellulose, pectin polysaccharide, etc.) is first enzymatically decomposed into single cells, and then the cell wall containing cellulose as a main component is decomposed by an enzymatic treatment such as cellulase. These two steps of enzyme treatment can be performed simultaneously.

[0008] The protoplasts thus isolated and prepared are also cultured by a known method. In the present invention, a hydrophobized polysaccharide is added to the medium. The hydrophobized polysaccharide is, for example, a natural polysaccharide such as pullulan, amylose, mannan, amylopectin, dextran, and xyloglucan phosphate modified with a hydrophobic group such as cholesterol or palmitoyl. , The above-mentioned natural polysaccharide and a hydrophobic group can be self-assembled in an aqueous solution (Macromolec
ules, 25, 5665-5670, 1992; Physicochemical Charact
erization of Cholesterol-Bearing Polysaccharides in
Solution: Organized Solutionsedited by Friberg,
SE and Lindman, B., Marcel Dekker. Inc., 290-30
4,1992). As an example of such a hydrophobized polysaccharide, the chemical formula of pullulan modified with a cholesterol group is shown below.

[0009]

Embedded image

By the method as described above, the cell membrane of protoplasts can be coated with the hydrophobized polysaccharide (see FIG. 1), which makes it possible to manipulate the originally unstable protoplasts in an extremely stable state. For example, normal protoplasts are calcium ionophore A23
It will die in a short time in the presence of 187 (Plant Science, 69,
135-138, 1990), the artificial cell wall-coated protoplasts stabilized by the method of the present invention show high resistance to A23187 as shown in the following examples.

Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to the following examples.

[0012]

Example 1 (Preparation of carrot protoplasts and their stability
Reduction) was first prepared protoplasts by the following procedure from carrot cells. Carrot suspension culture cells of the material is derived from a carrot cultivar Kuroda-gos u n, Murasige - Skoog medium [saccharose 3%, 2,4 - dichlorophenoxy acetic acid (2,4
D) 1 mg / L, containing kinetin 0.1 mg / L: pH was adjusted to 5.8 before autoclaving) and subcultured. Transplanting is performed every two weeks, 120 round trips per minute, amplitude 5 to 6c
The cells were cultured at 24 ° C. The cells on the 6th day after subculture were collected by filtration in a clean bench, and Murasige - Skoog medium (containing 0.25M mannitol and 0.25M sorbitol: pH
After washing once with 5.6), it was collected by filtration in a sterilized Erlenmeyer flask. Enzyme solution (Cellulase Onozuka R 10: 1.8% and D
riselase: retrieve the sterile filter 0.2% in tip-containing organic)
It was filled in with a syringe and added to enzyme solution in Erlenmeyer flask with Filtration sterilization. After sealing the flask, it was placed in a rotary infiltrator and allowed to infiltrate at 24 ° C. for 18 hours in the dark. The sample dispersion was filtered through a nylon filter with a pore size of 40 μm, the filtrate was centrifuged at 50 g for 5 minutes, the supernatant was removed, and the dispersion was redispersed with a washing solution, and then over 6% Ficoll sucrose solution (0.5 M). Was gently added to the cells and centrifuged at 200 xg for 5 minutes. Protoplasts agglomerated at the two-phase interface. The supernatant was removed, protoplasts were placed in a centrifuge tube with a capacity of 10 ml, dispersed with a washing solution, and centrifuged at 50 × g for 5 minutes. This operation was repeated 3 times. Finally, a small amount of protoplast culture medium (0.4 M mannitol added to the above cell culture medium) was dispersed to prepare carrot cell protoplasts.

Next, cholesterol group-modified pullulan (CHP) or xyloglucan phosphoric acid (CH XP ) was added to this protoplast dispersion at a rate of 0.3 mg / ml, respectively, and the mixture was added at 1 to 24 ° C. Shake gently for 3 hours
And they were cultured in the dark. Further, as a control, it was cultured protoplasts in the same manner even culture medium not containing CHP and CH XP.

The number of each protoplast cultured as described above was measured with a fluorescence microscope by the Fluorescein diacetate method, and the survival rate of each was determined. The results are shown in Table 1.
A are as indicated, protoplasts were cultured in CHP or CH XP-containing medium showed high <br/> had survival rate Izure compared with the control. In addition, the cholesterol is replaced
No pullulan (CHP) or xyloglucan phosphate
With (CHXP), effective coating and survival rate are all improved.
Was not recognized.

[0015]

[Table 1]

Furthermore, the state of the CHP coating is changed to Fluorescein.
CHP modified with isothiocyanate (CHP-FIT
Confocal fluorescence microscope (manufactured by Bio-Rad ) using C).
Observed at. That is, CHP-FITC is 0.6 mg /
Protoplasts were cultured in the dark at 24 ° C. for 3 hours in a protoplast culture medium added at a rate of ml. The result is shown in Figure 2.
It is as shown in the microscope photograph of only cell membranes CH
It has been confirmed clear that by the P-FITC has been overturned under. Example 2 (Stabilization test against A23187) CHP coated protoplasts and C prepared in Example 1
For H XP coated protoplasts were tested for resistance against calcium ionophore A23187.

The results are as shown in FIGS. 3 and 4. Note that FIG. 3 shows coated protoplasts (CHP, CH X in a medium containing A23187 at a concentration of 20 μM).
P) over time to indicate the viability of the control (uncoated protoplasts), 4 showed a survival rate after 2 hours incubation at each concentration of A23187. As apparent from these results, the artificial cell wall coating protoplasts coated cell membranes with hydrophobic polysaccharide by this invention method showed a much higher survival rate than the control group uncoated. Example 3 (resistance to intracellular influx of extracellular calcium
Test) The CH XP- coated protoplasts prepared in Example 1 were tested for resistance to calcium influx in the presence of A23187.

The results are as shown in FIGS. 5 and 6.
5 shows the survival rate of each protoplast after 2 hours of culture in a medium containing A23187 (20 μM) and calcium ions (0 to 10 mM), and FIG. 6 shows A2318.
7 (20 μM) and polysaccharides (0.0-1.2 / mg / ml)
Of each protoplast after 2 hours of culture in a medium containing
The survival rate is shown.

As is clear from these results, the artificial cell wall-coated protoplasts of the present invention showed superior resistance to extracellular influx of calcium as compared with the uncoated control group . Example 4 (Test for resistance to cellulase in culture solution) The CHP-coated protoplasts prepared in Example 1 were tested for resistance to cellulase, which is a cellulolytic enzyme.

Carrot protoplasts were cultured in the same protoplast culture solution (containing 0.3 mg / ml of CHP) as in Example 1 for 3 hours at 24 ° C. in the dark, and then cellulase (ONOZUKA R-10) was added at 0.3 mg / ml. It was added to the culture medium at a ratio of ml. After culturing for a certain period of time in this state, F
The survival rate of protoplasts was measured by a fluorescence microscope by the luorescein diacetate method. As a control, the protoplasts cultured in the CHP-free culture medium were also measured for survival rate in the cellulase-containing culture medium.

The results are shown in Table 2, and it was confirmed that the artificial cell wall-coated protoplasts of the present invention have high stability even in the presence of the cellulolytic enzyme cellulase.

[0022]

[Table 2] In addition, the addition of exogenous polysaccharide to protoplasts is generally
Improves the deposit of neoplastic cell walls on the original cell wall.
The same effect can be obtained with the artificial cell wall in this invention.
As a result of investigating whether or not
It was also found that it did not affect the birth of cellulose in the strike at all
did. In addition, DNCB (2,4-dichlorobenzonitril
e) inhibits cell wall regeneration in plant protoplasts
It However, the artificial cell wall coated protoplasts of this invention
So even in the presence of DNCB (12 μM)
Cellulose produced and regenerated was 6 mM EGTA
It was easily peeled off and the protoplasts were regenerated.

[0023]

As described in detail above, according to the present invention, it becomes possible to stabilize protoplasts which are originally extremely unstable with respect to calcium influx and cell wall degrading enzymes. The application range of protoplasts in the fields of engineering and biotechnology will be greatly expanded.

[Brief description of drawings]

FIG. 1 is a schematic view illustrating the method for stabilizing protoplasts according to the present invention.

FIG. 2 is a fluorescence micrograph showing CHP coating a cell membrane of an artificial cell wall-coated protoplast of the present invention.

FIG. 3: Artificial cell wall coated protoplasts (CHP, CHX) in medium containing A23187 at a concentration of 20 μM.
-P) and control ( uncoated protoplasts ) viability (%)
Shows the change with time.

FIG. 4 shows the concentration of A23187 after culturing for 2 hours.
Protoplast viability is shown.

FIG. 5 shows the survival rate of each protoplast after 2 hours of culture in a medium containing A23187 (20 μM) and calcium ions (0 to 10 mM).

FIG. 6 A23187 (20 μM) and polysaccharides (0.0-
The viability of each protoplast after 2 hours of culture in a medium containing 1.2 / mg / ml) is shown. ─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] April 6, 1995

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 3

[Correction method] Change

[Correction content]

[Figure 3]

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Name of item to be corrected] Fig. 4

[Correction method] Change

[Correction content]

[Figure 4]

[Procedure 3]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 5

[Correction method] Change

[Correction content]

[Figure 5]

[Procedure amendment 4]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 6

[Correction method] Change

[Correction content]

[Figure 6]

Claims (8)

[Claims] 1. A method for stabilizing protoplasts, which comprises culturing protoplasts in a medium containing a hydrophobic polysaccharide and coating the cell membrane thereof with the hydrophobic polysaccharide. 2. The method for stabilizing protoplasts according to claim 1, wherein the hydrophobized polysaccharide is pullulan, amylose, mannan, amylopectin, dextran or xyloglucan phosphate, which is partially hydrophobized. 3. The method for stabilizing protoplasts according to claim 1, wherein the protoplasts are plant cell protoplasts. 4. The method for stabilizing protoplasts according to claim 3, wherein the plant protoplasts are carrot protoplasts. 5. An artificial cell wall-coated protoplast in which the cell membrane is coated with a hydrophobized polysaccharide. 6. The artificial cell wall protoplast of claim 5, wherein the hydrophobized polysaccharide is partially hydrophobized pullulan amylose, mannan, amylopectin, dextran or xyloglucan phosphate. 7. The artificial cell wall-coated protoplast according to claim 5, wherein the protoplast is a plant cell protoplast. 8. A carrot protoplast whose cell membrane is coated with partially hydrophobized pullulan or xyloglucan phosphate.