JPS59216589A - Method for promoting fusion of plant protoplast - Google Patents

Abstract

PURPOSE:To fuse hybrid cells artificially from plant protoplasts and obtain efficiently useful hybrids easily and artificially from plant cells with a high probability, by using plant protoplasts treated with a fusion agent in the presence of a water-soluble aprotic polar compound under low osmotic pressure conditions. CONSTITUTION:Plant protoplasts, e.g. a protoplast such as a gromwell or carnation, cultivated in a liquid culture medium are treated with a fusion agent consisting of an aqueous solution containing polyethylene glycol and an alkaline calcium chloride, etc. and then allowed to stand in the presence of a water- soluble aprotic polar compound, e.g. dimethyl sulfoxide, under low osmotic pressure conditions to promote the fusion. Thus, the aimed fused cells of the plant protoplasts are obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of promoting fusion of plant protoplasts.

Rapid advances in tissue culture technology have led to attempts at cell fusion technology, which involves artificially fusing plant cells to grow new hybrid cells. There are also expectations for its creation. Fused cells obtained by cell fusion technology are used for various purposes that take advantage of their newly formed properties. For example, it is expected that useful secondary metabolites will be efficiently produced by mass culture, and certain A species will be used widely in agriculture and industry as new plants by differentiating organs from fused cells.

Cell fusion also uses plant cells (protoplasts) whose cell walls have been removed using enzymes, mechanical means, etc. However, simply mixing protoplasts has a low probability of fusion, so fusion has not been attempted in the past. Several methods to promote this will be tried and discussed.

Among these, the most excellent method is to treat protoplasts with a coalescing agent that is a combination of polyethylene glycol and an alkaline group 11Z calcium aqueous solution. Mainly, to further improve the process, it is also known that during the fusion process, operations such as: ■ increasing the temperature, ■ adding agents that disrupt cell membrane lining proteins, and ■ centrifuging can be used to promote protoplast fusion. It is being

However, although traditional methods improve the fusion rate somewhat,
This is still not sufficient, and in some plants, tA hardly fuses at all depending on the type of plant, and there has been a strong desire to improve this.

On the other hand, in order to maintain the stable existence of protoplasts, it is necessary to increase the osmotic pressure of the liquid holding the protoplasts. Protoplasts are maintained in a water bath containing sugar alcohols, inorganic salts such as calcium chloride, potassium chloride, and others.

Among them, the osmotic pressure of the aqueous solution holding protoplasts is about 7.
JoKy/cnH1 more preferably PJl
In addition, when treating protoplasts with a fusing agent, it is desirable that the range be from about 10 to about 100 m/C. Wash several times with water bath solution containing +7″1
After being flushed away, the protoplasts are kept in an aqueous solution containing an osmotic pressure regulator to maintain stability, and an environment that would destroy the protoplasts is avoided as much as possible.

In contrast, the present inventors have found that fusion is promoted by placing plant protoplasts that have been treated with a fusion agent under low osmotic pressure conditions, and based on this finding, using a fusion agent to produce plant protoplasts. We have invented a method for promoting the fusion of plant protoplasts, which is characterized in that the plant protoplasts treated with a fusing agent are placed under low osmotic pressure conditions. ).

On the other hand, plant protoplasts usually have an osmotic pressure of about /o
It cannot be maintained stably for a long period in a water bath solution with a Ky/c lower than the shoulder, and is particularly susceptible to destruction under osmotic pressure conditions as low as about 9 Ky/7.

Therefore, in the above method, it is necessary to avoid leaving the plant protoplasts treated with a fusion agent under low osmotic pressure conditions for a long time, and the time period for which they are left in these conditions is adjusted depending on the level of osmotic pressure. It is necessary to terminate the treatment before the plant protoplasts are destroyed.

That is, in a preferred embodiment of the above method, the plant protoplasts treated with the fusing agent are subjected to low osmotic pressure conditions, which are difficult to maintain stably for a long period of time, for a short period of time, for example, at an osmotic pressure of about i o My.
/cnz or less, especially about i o Ky /(yj A
It is left under the low osmotic pressure conditions of Fry/cA for about 5 minutes to about 43 hours, more preferably about 70 minutes to about 1 hour.

Furthermore, in the above method, the plant protoplasts treated with the fusing agent may be placed under low osmotic pressure conditions by adding a water bath solution, water, etc. with low osmotic pressure during the night of a water bath containing the fusing agent, or The water bath solution containing the agent may be replaced with an aqueous solution, water, etc. with low osmotic pressure.

Although the above method has the excellent effect of promoting the fusion of plant protoplasts, the present inventors have investigated methods for further promoting fusion and have found that plant protoplasts are grown under low osmotic conditions (sometimes It was discovered that fusion is further promoted by the coexistence of a water-bathable aprotic polar compound, and the present invention was developed.In other words, the present invention provides a method for fusing plant protoplasts using a fusing agent. A method for promoting fusion of plant protoplasts, which is characterized in that plant protoplasts treated with an agent are fused under low osmotic pressure conditions in the coexistence of a water-soluble aprotic polar compound. They are not particularly limited (for example, medicinal herbs such as violet and ginseng, garden plants such as carnations and roses, vegetables such as cabbage and eggplant, and grains such as rice and wheat)
The present invention is applicable to cell fusion between plants of the same species or between plants of different species.

In the present invention, plant protoplasts are prepared from parts of plants or cultured cells using enzymes such as cellulase and pectinase, or by mechanical means. , fusion treatment is carried out by adding a fusion agent to a water bath solution containing plant protoplasts, or replacing components other than plant protoplasts in a water bath solution containing plant protoplasts with a fusion agent or an aqueous solution thereof. The operation can be divided into several steps and performed step by step, if necessary.

As a fusing agent, for example, polyethylene glycol (PE
G), polypropylene glycol (PPG).

Synthetic polymers such as polyvinyl alcohol (PVA), natural polymers such as dextran and pectin, and inorganic salts such as calcium salts and sodium nitrate are used. These fusing agents may be used alone or in combination of one or more types as necessary, but among them, PEG, P
VA and dextran are preferred, especially PEG. PEG with an average molecular weight of about 1 soo to about 7,300 is suitable, and it is desirable to use it as a water bath solution with a PEG concentration of about JO to about 70 W/W%. It is placed in an environment of a water bath containing a coalescing agent. At that time, an aqueous solution containing a coalescing agent with a relatively high osmotic pressure is used;
Appropriate adjustments are made so as not to adversely affect the retention of plant protoplasts.In the case of fusion treatment with a fusion agent, the osmotic pressure is adjusted to For this reason, the osmotic pressure of the aqueous solution containing the fusing agent may be adjusted with an osmotic pressure regulator, if necessary.The osmotic pressure regulator may include the above-mentioned saccharides, sugar alcohols, Examples include inorganic salts.

Plant protoplasts treated with a coalescing agent are placed in hypoosmotic conditions in the presence of a water-bathable aprotic polar compound.

In this case, a preferred embodiment is to subject the plant protoplasts treated with the fusing agent to low osmotic pressure conditions, which are difficult to maintain stably for a long period of time, for a short period of time, for example, at an osmotic pressure of about IO[7/ctl or less, especially about io My/ctA to 211y,
/ About 3 minutes to about 3 hours under the low osmotic pressure condition of the shoulder,
Further □ is preferably about 10 minutes to about 2 hours.

As a means of placing plant protoplasts treated with a coalescing agent in a low osmotic pressure condition in the coexistence of a water-soluble aprotic polar compound, a water-soluble aprotic polar compound is added to an aqueous solution containing the coalescing agent and plant protoplasts. Although there is a method of directly adding the compound, it is preferable to add a water bath solution of low osmotic pressure containing a water-soluble aprotic polar compound, or to add a part or all of the water bath solution containing the coalescent and plant protoplasts to water. An example is a method of replacing the water with a low osmotic pressure water bath solution containing an aprotic polar compound. In the present invention, a low osmotic pressure water bath solution to which a water bath aprotic polar compound is added is used. It is desirable to add to this low osmotic water bath solution, as long as the low osmotic pressure conditions are maintained, components that are sometimes used as conventional culture medium components for normal plant cell culture. Examples of such ingredients include sugars such as sucrose and glucose, sugar alcohols such as mannitol and sorbyl, and calcium chloride.

There are inorganic salts such as magnesium salts, and these can be used alone or in a suitable combination of one or more of them. Among these, saccharides and inorganic salts are preferred, and calcium chloride and calcium chloride and saccharides are preferably used in combination.

When an aqueous solution with low osmotic pressure is used, a small number (seven or more types) of compounds selected from water-soluble aprotic polar compounds are added.

Examples of the aprotic polar compounds in the aqueous solution used in the present invention include 1, sulfones such as sulfolane and dimethylsulfone, sulfoxides such as dimethyl sulfoxide and diethyl sulfoxide; N,N-dimethylformamide, N,N-diethylformamide; N,
N-dimethylacetamide, N-methylpyrrolidone, N
-Ethylpyrrolidone, N-Inglovirpyrrolidone, N
-Octylpyrrolidone, N-cyclohexylpyrrolidone, N-benzylpyrrolidone, polyvinylpyrrolidone, N
Amides such as -methyl-2-pyridone and uN-methyloxazolidone; phosphoric triamides such as hexamethyl phosphoric triamide and hexaethyl phosphoric triamide; N,N,N',N'-tetramethylurea s
Examples include substituted ureas such as 'l'l dithi-2-imidazolitone. Among these, dimethylformamide, dimethylsulfoxide or dimethylacetamide are preferred.

In the present invention, the aprotic polar compound may be used alone, or two or more types may be used in combination.

In the present invention, the ratio of coexisting aprotic polar compounds is determined by adding a low osmotic water bath solution containing an aprotic polar compound, or replacing a part or all of a low osmotic water bath solution containing plant protoplasts. Approximately 0.71/00 ml of aprotic polar compound per 71/00 volume plate. If the tray portion has a capacity of 1 to 10 liters, the effect of promoting fusion tends to decrease.

In the present invention, the amount of the aqueous solution with low osmotic pressure to be used can be changed as appropriate depending on the desired low osmotic pressure conditions and the concentration of the above-mentioned specific components to be coexisting. For example, in an embodiment in which a low osmotic aqueous solution is added to an aqueous solution containing a coalescing agent, the low osmolarity aqueous solution is added in an amount of about 1 to about 704 parts by volume per 1 volume part of the aqueous solution containing a coalescing agent, and immediately about IO to about 704 parts by volume of the aqueous solution containing a coalescent. Approximately 703 parts by volume is added (preferably) under the desired hypotonic pressure conditions.

In the practice of the present invention, other known fusion-promoting methods may be used, such as high temperature treatment (about JO to about 5o72), denaturants of cell membrane lining proteins (e.g., cytochalasin B
, N-ethylmaleimide] treatment, centrifugation treatment (approximately qO
)7. C) can also be used in combination.

According to the present invention, by placing plant protoplasts in a bag under low osmotic pressure conditions in which the above-mentioned specific components coexist, fusion can be further promoted by placing plant protoplasts under low osmotic pressure conditions (compared to the case where plant solidoplasts are simply placed under low osmotic pressure conditions). .

Furthermore, the fused protoplasts obtained by the method of the present invention are
They can be isolated by conventionally known methods, or they can be cultured in a medium with an osmotic pressure suitable for maintaining fused plant protoplasts to produce hybrid cells with regenerated cell walls, which can then be separated and used by conventionally known separation methods. .

Examples of the present invention are shown below.

Example 1 Lithospermune cultured in a liquid medium of Linsmeyer-Skoog (/91.!; year).
r)'throrhizon 5eib, et Zu
cc,) cultured cells were added with glucose to increase the osmotic pressure to /
Protoplasts were obtained by treatment for 10C1λ hours in a cell wall degrading enzyme water bath solution with the following composition adjusted to /, Ky /7.

Composition of cell wall degrading enzyme water bath solution 4 Shichinoze “Onozuka’fl-10 (manufactured by Kinki Yakult) ζθ Section Driselase (manufactured by Kyowa Hakko) O,
SS Macerozyme R-10 (manufactured by Kinki Yakult) 0
．． 6M/A Glucose 3mM/lk Calcium chloride! rmM/A Magnesium chloride The protoplasts obtained as above were
/ ci glucose aqueous solution (0, m/a glucose, km m/z calcium chloride, 3 m m/a
After suspending the protoplasts in magnesium chloride to adjust the density of the protoplasts to CO x IO6 cells/1 nb, this suspension, 0 μk, was dropped onto a Petri dish made by Falcon, Inc. and having a diameter of 1.5 mm. Polyethylene glycol aqueous solution of -20μ after standing for S minutes cso W/W4 polyethylene glycol q000, j OmM/A calcium chloride, sso
mm/I! sglucose] was added and allowed to stand for 10 minutes.

then pH/0 at an osmolality of 9 Kf/cnl,
! alkaline calcium chloride water bath 1 & (50mM
/ A salt 1 and calcium, 50mM/! glucose)
/, +++A was added at a rate of 0.2 mb/min (osmotic pressure is approximately 9'j'/cdt) and then left to stand for 10 minutes.

When the fused protoplasts were counted under a microscope, the percentage of fused protoplasts (cell fusion rate) was
Tl1j was 74.

Comparative Example 1 In Example 1, alkaline salt 1 with an osmotic pressure of 9 El/cm containing dimethyl sulfoxide of /(V/V) 4
instead of the same calcium water bath solution (pH/0, k).

Osmolality 9KP/c without dimethyl sulfoxide
A alkaline calcium chloride aqueous solution 1(1)H/17.
The same procedure as in Example 1 was carried out except that 5) was used.

As a result, the cell fusion rate was 30.

Comparative Example 2 Protoplasts obtained in the same manner as in Example 1 were incubated at an osmotic pressure of 13
After adjusting the density of protoplasts by suspending them in an aqueous glucose solution (0, ≦M/!glucose, 5mM/A calcium chloride, SmM/!magnesium chloride) of 50%/ct7, this Suspension -
〇μk was dropped onto a Petri dish made by Falcon, Inc. and having a diameter of 5C1n. 10μ after standing for S minutes! Polyethylene glycol water bath i (30W/W'lb polyethylene glycol q00θ, left OmM/A calcium chloride, coS
θmM/A glucose) was added and allowed to stand for 10 minutes.

Then, at osmotic pressure l<tKy/cm, pH/0,5,
/ (V/V) 1 of an alkaline calcium chloride aqueous solution containing dimethyl sulfoxide (50mM/A calcium chloride, 0mM/A glucose) and 0.λ
After adding mA at a rate of 7 minutes (osmotic pressure is approx./1 lKy)
/cst and left standing for 19 minutes.

When the fused protoplasts were counted under a microscope, the percentage of fused protoplasts (cell fusion rate) was 1/1.

Comparative Example 3 In Comparative Example 2, instead of the alkaline calcium chloride aqueous solution (pH 10, t) with an osmotic pressure of la Ky / cM containing dimethyl sulfoxide of / (V/V), an alkaline solution with an osmotic pressure of / IA Kg / crux was used. The same procedure as in Comparative Example 2 was carried out except that 1 g of calcium chloride aqueous solution (pH 10, j) was used.

As a result, the a-cell fusion rate was 3%.

Comparative Example 4 In Comparative Example 2, dimethyl sulfoxide was
) Comparative Example 2 was carried out in the same manner as in Comparative Example 2, except that dimethylformamide was used at a ratio of /(V/V)% instead of using a ratio of 1%.

As a result, the cell fusion rate was 9.

Example 2 Tobacco (N1cotiana taba) grown in a greenhouse
Toshiyuki Nagata and Itaru Takebe used mesophyll cells of P. cumL, var, and Samsun to
Protoplasts were prepared according to the method reported in NTA Monthly Volume 99, 1971, Page 7λ.

The protoplasts thus obtained were osmoticed,
31ij'/cm mannitol water bath solution (o, s
M/! After adjusting the density of protoplasts to 10 cells/mb by suspending them in mannitol, 5 mM/A salt, calcium, 3 mM/j magnesium chloride,
This suspension is 30μ! was dropped onto a Falcon Petri dish with a diameter Sα. After standing still for 10 minutes, add polyethylene glycol aqueous solution C70W/W4 polyethylene glycol isao, iomM/b salt qt calcium, / x OmM/A glucose) to JOμ and incubate with lS.
It was left standing for a minute.

Rum aqueous solution (!r OmM/A calcium chloride, 5
After adding 0mM/7glucose) pib at a rate of o, 3m67min (osmolarity is approximately 9 hp/ctl), /,! It was allowed to stand for 1 minute.

When the fused protoplasts were counted under a microscope, the percentage of fused protoplasts (cell fusion rate) was to%.

Comparative Example 5 In Example 2, the osmotic pressure solution containing 1 (V/V)% dimethyl sulfoxide was PH/0 at Kf/ctii.
, ! Except for treating with an alkaline calcium chloride aqueous solution (pH/0, k) with an osmotic pressure of 9 KP/ca instead of the alkaline calcium chloride aqueous solution;
The same procedure as in Example 2 was carried out.

As a result, the cell fusion rate was Fi'104.

Example 3 Murasaki (Lithospermumeryt) cultured in a liquid medium of Linsmeyer-Skoog (/9/S)
hrorhizon 5eib, et Zucc,
) and carnation (Dianthus Cary
cultured cells of P. ophyllus L.) were incubated in the same cell wall degrading enzyme water bath solution used in Example 1 for 30 min each.
After treatment for 0.013 hours, protoplasts were obtained.

The protoplasts obtained in this way were each given an osmotic pressure of 'lKf/ct! glucose aqueous solution (0.6M/
A glucose! r mM/A calcium chloride, S
After mixing equal amounts of protoplasts suspended in [mM/A magnesium chloride] and adjusting the density of protoplasts to 106 cells/mb, this suspension -〇μk was divided into a diameter of 1tffl.
After dropping it on a Falcon Petri dish for 05 minutes, a 10 μm solution of polyethylene glycol (1
! -OW/W qb polyethylene glycol group 000,
s o mM/A salt 1 and calcium, -1 somM/
A glucose] was added and allowed to stand for 10 minutes.

Then dimethylformamide /,, o (V/V
) and osmotic pressure t [j'/cnr in pH/
0,5 alkaline calcium chloride water bath solution (!;
OmM/A calcium chloride, s o mM/4glucose] 6ml Wo, J.

After adding mb at a rate of 7 minutes (osmotic pressure is approximately JK/
cnl] and allowed to stand for 10 minutes.

(The protoplasts in which the purple protoplasts and carnation protoplasts were fused were counted under a microscope, and the percentage of fused protoplasts (i. vacuole fusion rate) was 2%.

Claims (1)

[Claims] A method for fusing plant protoplasts using a fusing agent, which comprises fusing plant protoplasts treated with a fusing agent under low osmotic pressure conditions in the coexistence of a water-soluble aprotic polar compound. Fusion promotion method 0