JPH04252178A - Culture of plant cell - Google Patents

Abstract

PURPOSE:To carry out culture of plant cell capable of efficiently maintaining and multiplying plant cells having redifferentiating capacity by culturing plant cells while controlling pH of liquid medium in a fixed range with an ammonium ion-containing alkali, etc. CONSTITUTION:In culture of plant cell of rice plant or carrot with a liquid medium, the plant cells can be cultured while controlling the liquid medium in a fixed range of pH 5.7-5.9 with an ammonium ion-containing alkali such as ammonia water and a nitrate ion-containing acid. In the first half of the culture, ammonium ion is maintained approximately constant and in the second half, nitrate ion is preferably kept approximately constant.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a plant cell culture method, and more particularly to a plant cell culture method that can efficiently maintain and propagate plant cells with regeneration potential in liquid culture.

0002

[Prior art] In plant cell culture, the pH of the medium
, ammonium ion, and nitrate ion are determined at the time of culture medium preparation, and are generally not controlled during culture. In plant cell culture, these three have very important roles, and their optimum values or optimum concentrations are variously studied as values before culturing and determined for each plant cell. Also,
Medium pH may be controlled during culture, with the purpose of correcting pH changes caused by plant cells ingesting inorganic ions in the medium during culture. In this case, adding a buffer to the medium can be used instead.

However, the conventional plant cell culture method described above has a problem that needs to be improved in terms of the efficiency of plant cell proliferation.

0004

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for culturing plant cells that improves the efficiency of plant cell proliferation.

[0005]

[Means for Solving the Problems] As a result of intensive research to solve the above problems, the present inventor has developed a method for culturing plant cells using a liquid medium, in which the pH of the liquid medium is adjusted to an alkali containing ammonium ions and an acid containing nitrate ions. We have discovered that by culturing plant cells while controlling them within a certain range, the growth efficiency of plant cells can be significantly improved while maintaining their redifferentiation ability, and based on this new knowledge, we have completed the present invention. .

[0006] Therefore, the plant cell culture method of the present invention
In the plant cell culture method using a liquid medium,
Maintaining plant cells with regeneration ability by culturing plant cells while controlling the pH within a certain range with an alkali containing ammonium ions and an acid containing nitrate ions.
It is characterized by being allowed to proliferate. As a specific example of controlling the pH of the liquid medium to a certain range using an alkali containing ammonium ions and an acid containing nitrate ions, in the first half of culturing plant cells, ammonium ions in the liquid medium are kept almost constant. In the second half, the nitrate ions are held almost constant.

Examples of plants to which the present invention can be applied include rice, carrots, parsley, celery, soybeans, melons, garlic, and asparagus. The liquid medium used for liquid culture of plant cells may be any liquid medium that can be used for normal plant cell culture, but preferably MS medium,
N6 medium, SH medium, LS medium, white medium, B5
Examples include culture medium and the like.

[0008] The above-mentioned plant cells can be obtained by various methods, but for example, plant explants such as growing points, hypocotyls, leaves, roots, etc. are treated with a carbon source, an inorganic salt, and a plant hormone such as auxin. It can be obtained by subculturing only the amorphous cell mass generated from the explant in a similar medium, for example, using a container such as an Erlenmeyer flask or a jar fermenter.

[0009] An alkali containing ammonium ion is
Any alkali that contains ammonium ions and has a high pH may be used; for example, aqueous ammonia can be used. The acid containing nitrate ions may be any acid containing nitrate ions and having a low pH, such as nitric acid.

[0010] The above pH control is performed by adjusting the pH of the medium to p
It is measured by an H sensor, and when the pH of the culture medium falls below a preset lower limit, it is added by an alkaline pump containing ammonium ions, and when the pH of the culture medium exceeds the lower limit, the pump stops, and when the pH of the culture medium exceeds a preset upper limit, This can be done by adding an acid containing nitrate ions using a pump, and stopping the pump when the amount falls below the upper limit.

Having the ability to regenerate means that plant cells are placed in a medium containing a carbon source, inorganic salts, and plant hormones, and the plant cells are used to form a tissue that can become a plant in the future, such as an indeterminate tissue. It means that embryos, adventitious buds, embryoid bodies, etc. can be formed.

0012

[Function] As described above, according to the present invention, an alkali containing ammonium ions and an acid containing nitrate ions cause
By controlling the pH of the medium within a certain range and culturing plant cells in liquid, plant cells with regeneration ability can be maintained and grown with higher growth efficiency than conventional methods.

[0013]

[Example] Rice cells induced from seed scutellum and subcultured were placed in an aerated agitated culture tank containing fresh medium for 55 minutes.
g/L, and after culturing for 2 weeks, the fresh weight of the cells was measured. During the culture, the pH of the medium was controlled to 5.7 to 5.9 using 5% ammonia water and 1N nitric acid.

The experiment was carried out three times. The results are shown in Table 1.

[0015]

[Table 1]

(Note) In Table 1, the proliferation rate is defined as b/, where ag is the amount of cells inoculated at the start of culture (fresh weight), and bg is the amount of cells (fresh weight) at the end of culture.
Represented by a. In both experiments, the conventional culture method (
The growth rate was 1.86 (average) in the culture method according to the present invention, when the growth rate was 1 in the conventional culture method. Furthermore, the regeneration ability was superior to that achieved by conventional culture methods.

[0017] When ammonium ions and nitrate ions in the medium were analyzed using high-performance liquid chromatography and their fluctuations were investigated, it was found that before a certain point, ammonium ions and nitrate ions changed in the medium. It was under constant control. (Figure) The regeneration ability was evaluated by placing rice cell clusters on a germination medium and counting the number of germinations formed. The results are shown in Table 2.

[0018]

[Table 2]

(Note) In Table 2, the germination rate is expressed as b/a, where a is the number of cell clusters placed on the bed, and b is the number of cell clusters in which germinated bodies are formed after culturing. Next, the medium composition and culture method used are shown. <Cell culture conditions> 500m containing 250ml of medium
In a 1 volume aerated stirring type culture tank, 25-27℃, 50rp.
Cultured at m. Alternatively, culture at 30°C and 50 rpm in a 2L aerated agitation culture tank containing 1L of medium. <Conditions for inducing germinants> A total of 80 cell clusters of 1 mm or more are placed in a 9 cm Petri dish, 16 cells per Petri dish. Culture at 25-27°C for 4 weeks.

[0020]

Claims (2)

[Claims] Claim 1: In a method for culturing plant cells using a liquid medium, the pH of the liquid medium is controlled within a certain range using an alkali containing ammonium ions and an acid containing nitrate ions, and plant cells are cultured to have regeneration ability. A plant cell culture method characterized by maintaining and propagating plant cells. 2. The method according to claim 1, wherein ammonium ions in the liquid medium are kept almost constant during the first half of culturing the plant cells, and nitrate ions are kept almost constant during the second half. Plant cell culture method.