JPS6255077A - Method for plant cell culture - Google Patents

Abstract

PURPOSE:To enable the division and proliferation of plant cell, especially a protoplast in high efficiency, by including and immobilizing a plant cell in a spherical gel containing a polysachharide produced by a microbial strain belonging to Pseudomonas genus and culturing the immobilized plant cell. CONSTITUTION:A plant cell containing protoplast and spheroplast is included and immobilized in a spherical gel having a diameter of 1.0-5.0mm and containing 0.2-0.6wt% substance obtained by deacetylating a polysachharide produced by Pseudomonas elodea and secreted from the cell and purifying the deacetylated polysaccharide, and the immobilized cell is cultured.

Description

[Detailed Description of the Invention] [Industrial Application Field] This invention is a plant cell obtained by deacetylating polysaccharides produced by Pseudomonas Elodea and secreted outside the bacterial cells, and then purified. This invention relates to a method of encapsulating and immobilizing a substance in a spherical agglutinated body and then culturing it.

[Prior art and its problems] The production of useful substances by applying cell culture has traditionally been actively carried out using microorganisms and the like. In recent years, attempts have often been made to use plant cell culture for similar purposes, such as the production of plant alkaloids, steroids, and the like.

However, many problems still remain in achieving constant mJl of useful substances. That is, unlike microorganisms, they have a low growth ability, it is difficult to achieve a certain level of productivity, and it is difficult to cultivate them in large quantities.

Therefore, a method has been adopted for plant cells including protoplasts and spheroplasts, which are immobilized by embedding or encapsulating them in agglomerated bodies and then post-cultured. Recently, agglomerated polysaccharides derived from seaweed such as agar, agarose, alginate, and carrageenan have been used as carriers for such immobilization. However, when these substances are used, it is difficult to immobilize a certain number of cells into globuloids of a desired size, and even if they can be immobilized, it is not easy to efficiently divide and proliferate am. do not have. In any case, in order to fully develop plant cell engineering, which has a wide range of applications such as plant genetic manipulation, cell fusion, and substance production, it is necessary to efficiently divide and divide the plant cells, especially protoplasts, which are the basis of plant cell engineering. There is a lot to be gained in the development of methods for redifferentiation.

[Means for Solving the Problems] The present inventors have compared agglomerated bodies that promote the division and proliferation of plant cells, including many species of protoplasts and spheroplasts with varying degrees of cell walls. As a result of our investigation, we found that Pseudomonas aeodia (Ps
The polyMM produced by Eudomonas elodea and secreted outside the bacterial cell is purified after deacetylation. ? It was discovered that the agglomerated form of the substance used in this study has such an effect. In addition, as a result of examining the method of using the coagulant, it was found that a remarkable synergistic effect can be seen when the above-mentioned plant ivI is encapsulated in a coagulant formed into a sphere of a certain size, fixed, and then cultured. It was.

The plant cell culture method of the present invention utilizes the property that the above-mentioned substance can be instantly coagulated under the influence of magnesium ions contained in ordinary plant culture media (1 to 1.5 mM). Plant II vesicles such as protoplasts are suspended in a solution of a constant concentration of the above substance at a desired density, and a desired amount of the suspension is released into a liquid medium. The released suspension becomes spherical and instantly coagulates due to the action of magnesium ions in the medium. The size of the globuloids can be adjusted by changing the release fear, and the number of cells encapsulated in the globules can be easily adjusted by changing the cell density in the suspension. can do. Furthermore, the hardness of the globular aggregates can be easily adjusted by changing the concentration of the above-mentioned substances or by changing the magnesium ion concentration in the culture medium.

Since the concentration of the above-mentioned substance solution is independent of temperature, there is no need to subject the cells to high temperatures as is the case when conventional agglutinants such as agar, agarose, etc. are used.

Moreover, as a condition for the agglutination of the above-mentioned substance, it is sufficient that magnesium ions are present at a concentration contained in a normal culture medium. Furthermore, since the gelatinization occurs instantaneously, there is no need to suspend the cells in a solution containing a high concentration of calcium or potassium ions for a certain period of time, which is done when alginate and monocarrageenan are used as the gelatinant. Therefore, according to the method of the present invention, treatments that may damage cells (e.g., high temperature treatment, high concentration metal ion treatment, etc.) can be omitted, and the survival rate of cells can be significantly improved. In addition, cell division and proliferation can be extremely promoted.

As the culture medium for the method of this invention, any medium containing ordinary magnesium ions can be used. However, if the all cells to be cultured are protoplasts, D-7 nitol, D-sorbitol, glucose, sucrose, etc., which are commonly used for osmotic pressure adjustment, may be added to a constant concentration solution of the above substances and the culture medium. It is desirable to add it to

[Example] Example 1 Pseudomonas elodia (P seudomona
Using commercially available Gellan Gum (trademark) (Merck & Co., USA), which is a substance obtained by acetylating and purifying polysaccharides produced by S. elodea and secreted outside the bacterial cells, the agglutinated bodies were used to stimulate IIl cell proliferation. We investigated the effects of

As a comparative example of coagulant, sodium alginate (UK,
808), carrageenan (FMC, USA), agar (Difco, USA), and agarose (FMC, USA). tIAII21, which was the subject of investigation here, is a protoplast isolated from a suspension culture of Dice. All of the above coagulant substances were dispersed in a solution with the composition shown in Table 1 (however, in the case of carrageenan, 10 g/β of NaCl lx was added), and autoclaved at 121°C for 15 minutes. Sterilized.

Table 1 Composition of solution for preparing agglomerated bodies *2 -(N-morpholino)ethanesulfonic acid hydrate The protoplasts described above were encapsulated in the various types of agglomerated bodies described above as follows. When using gellan gum, first O9
The protoplasts were suspended in a 4x ω% gellan gum solution and injected into a micropipette. Next, the suspension was directly released into the culture solution shown in Table 2 to form spherical aggregates with a diameter of 2 m.

When using sodium alginate, first 4.0% by weight
The protoplasts were suspended in a sodium alginate solution and injected into a micropipette.

Next, the suspension was 7% by weight containing 50mM calcium chloride.
was added dropwise to the glucose solution. After being left at room temperature for 30 minutes, it became a sphere with a diameter of 2 m and solidified. 7% by weight of the spherical agglutinated body
After washing thoroughly with a glucose solution, the cells were transferred to a culture medium. - When using carrageenan, first incubate at 35°C.
The protoplasts were suspended in a solution of 1% by weight Guinan. Next, the suspension was poured into a Teflon plate with holes of 2.0 m in diameter, and after cooling, the suspension was poured into a Teflon plate with a diameter of 21 M1.
A cylindrical agglomerated body with a height of 1.3M was obtained. The columnar agglomerates were exposed to a 7% by weight glucose solution containing 300mM calcium chloride for 60 minutes, then thoroughly washed with a 7% by weight glucose solution and cultured! Moved to I'a.

When agar and agarose were used, the protoplasts were first sufficiently suspended in a 2% by weight agar solution kept at 40°C or a 2% by weight agarose solution kept at 35°C. Next, the suspension was poured into a Teflon plate with holes of 2.0 m+ in diameter and 2 m in diameter and 1.3 m in height.
A cylindrical agglutinated body was obtained. In either case, the same number of protoplasts, ie, 50 protoplasts, were encapsulated in the agglomerated body having approximately the same volume and weight of 41N.

Fifty spherical or cylindrical agglutinated bodies in which protoplasts were encapsulated and immobilized as described above were placed in plastic plates with a diameter of 5 cm, and cultured by adding 2 d of the medium shown in Table 2 below. The culture was carried out at 25° C. under 200 lux light and shaking at B□rpm. The survival rate of protoplasts immediately after isolation was 92%. In each case, three plates were prepared. After one month of culture, five spherical and cylindrical agglomerates were arbitrarily selected and the state of colony formation was observed under a microscope. The results are shown in Table 3.

Table 2 Composition of culture medium Table 3 Diagnosis of the protoplast division effect exerted by each agglutinated body Weight 1 M and solidified +4 weight number (5 o) Example 2 Gellan gum globular agglutinated bodies - The colony formation rate was examined by changing the number of 'protoplus' enclosed per individual. Preparation of the medium, culture method, and colony formation rate assay were performed according to Example 1. The protoplasts used here were protoplasts isolated from a suspension culture of Dice as in Example 1.

Encapsulation of protoplasts into gellan gum globular aggregates was carried out in the same manner as in Example 1, but the number of protoplasts per aggregate was changed to 50 ffl, 25 pieces, 10 pieces, and 5 weight 1. In testing the colony formation rate, 5 pieces, 10 pieces weighing 1.25 g, and 50 pieces of globoid agglomerates were taken out from the plates cultured at different protoplast densities as described above. The survival rate of protoplasts immediately after isolation was 94%. The results are shown in Table 4.

Table 4 Effect of protoplast density Example 3 The effect of the encapsulation method using gellan gum on cell proliferation was compared with that using agarose. here,
Cell clusters derived from tobacco mesophyll protoplasts were used. The protoplasts were statically cultured for one month at a density of 5×10′ cells/d in a liquid medium having the composition shown in Table 2. One month later, the culture was filtered using a nylon mesh with a diameter of 200 μm, and the cell mass (mainly a mass of I cells that had divided two or three times) captured on the mesh was obtained as a material. The cell mass was suspended in the solution shown in Table 1 containing 0.31% gellan gum, and the suspension was released into the culture medium. At this time, the release amount and cell mass density were adjusted so that the diameter of the resulting agglutinated body was 2 m and the number of cell clusters contained in the agglutinated body was 25. However, the medium used here had the glucose concentration in the composition shown in Table 2 as type 2 %. Other operations were performed according to Example 1. After 1 month of culture weight, the number of colonies whose layer ends had a diameter of one foot or more was counted, and the frequency thereof was determined.

The results are shown in Table 5.

Table 5 Effect of nizing agent on cell mass [Effect of the invention] According to the culture method of the present invention, plant cells such as protoplasts can be encapsulated and immobilized in agglomerated bodies, so that they can be protected from mechanical stimulation caused by shaking culture etc. It is possible to protect cells. In addition, Pseudomonas elodia (P 5eudon+o
When the polysaccharide produced by Nas elodea and secreted outside the bacterial body is encapsulated and immobilized in a spheroidal agglomerate of a substance obtained by deacetylating and then decontaminating, cell division and proliferation are significantly promoted. Ru. Such an effect will greatly encourage the redifferentiation of protoplasts into plants and the development of material production technology through plant cell culture.

Claims (4)

[Claims] (1) In culturing plant cells, the plant cells are cultured as Pseudomonas elodea (Pseudomonas elodea).
A substance obtained by deacetylating and purifying polysaccharides produced by B. odea) and secreted to the outside of the microbial cell is encapsulated in a spherical agglutinated body with a diameter of 1.0 to 5.0 mm containing 0.2 to 0.6% by weight. A plant cell culture method characterized by culturing after fixation. (2) The plant cell culture method according to claim 1, wherein the plant cell is a plant cell having a complete cell wall. (3) The plant cell culture method according to claim 1, wherein the plant cells are spheroplasts having various degrees of cell walls. (4) The plant cell culture method according to claim 1, wherein the plant cell is a protoplast completely lacking a cell wall.