JPS6236184A - Method of plant cell culture - Google Patents

Abstract

PURPOSE:To raise survial rate of plant cell and to raise division frequency, by adding a fixed amount of albumin to an arbitrary medium and cultivating a culture cell. CONSTITUTION:An albumin (bovine serum albumin, chicken glair albumin, lactoalbumin, lysine, leucosine, legumelin, etc.,) is added to an arbitrary medium and a plant cell is cultivated. Even in a case where a medium having a different component composition is used depending upon each cell seed, the survival rate of a plant cell containing complete protoplast of many seeds and spheroplast with a cell wall of various degrees can be raised and division is promoted.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for culturing plant cells using a medium supplemented with albumin.

[Prior art and its problems] In recent years, research on plant breeding and substance production using plant cells has been actively conducted. That is, am engineering techniques that create new varieties or produce target substances through cell fusion, or genetic engineering techniques that utilize plant vectors by microinjection of DNA, etc., are being put into practical use. However, a major obstacle to the development of such methods is that there is no well-established method for efficiently dividing and regenerating plant cells, especially protoplasts, after such manipulations. be. In particular, it has not yet been possible to regenerate individuals from protoplasts for many major agricultural crops. Generally, a certain degree of high cell density is required for the growth of plant cells in the culture stage, and the lower the cell density, the more difficult the growth becomes. From this point of view, there is a strong desire to establish a plant cell culture method that has high cell division frequency and survival rate and is repeatable.

[Means for Solving the Problems] The inventors have determined that the viability of plant cells, including complete protoplasts of many species and spheroplasts with varying degrees of cell walls, can be improved by simply adding them to a normal culture medium. As a result of searching for a substance that can increase the amount of albumin and promote division, it was discovered that various albumins have such an effect.

The plant cell culture method of the present invention is characterized by adding a certain amount of albumin to any medium for culturing. Albumin is a general term for a group of soluble proteins that are widely present in the tissues of animals and plants, and includes not only simple proteins but also complex proteins containing a small amount of sugar. Examples of such albumins include animal albumin such as bovine serum albumin, chicken egg albumin, lactalbumin, etc., and vegetable albumin such as lysine, leucosine, legumelin, etc. When various commercially available albumins were assayed, it was found that almost all albumins had the effect of significantly promoting cell survival rate and division frequency, although there were differences in degree. When using the albumin preparation, one with high albumin purity was used.

Such an effect of adding albumin is universally recognized regardless of whether the plant is woody, herbaceous, monocotyledonous, or dicotyledonous, and regardless of the plant tissue from which it is derived.

[Example] Example 1 Using protoplasts isolated from various plant materials,
The effect of adding albumin to the medium on the cell division frequency was investigated. The plant tissues from which protoplasts were directly prepared were all young seedlings whose seeds had been germinated aseptically, except that adult leaves were used by cutting. All of these protoplasts were grown in a medium with the composition shown in Table 1 (non-supplemented medium), and in the medium supplemented with 0.1% (w/v) bovine serum albumin (fraction V: Sigma, Missouri, USA). , St. Louis)
The cells were cultured in a medium supplemented with (supplemented medium). The medium was prepared as follows. First, two-fold concentration media were prepared, one without albumin and one containing 0.2% (W/V) albumin, and these were sterilized by filtration. Separately, 0.1% (W
A 0.8% (W/V) agar solution containing activated carbon (V) was sterilized by autoclaving and kept at 40° C. to prepare a solution. This agar solution and a medium having a double concentration were mixed at a volume ratio of 1:1, and the mixture was placed in 1 d portions into a 3G diameter plastic petri dish, and cooled and solidified. On this agar medium, 10μ℃ of protoplasts (approximately 100a in cell number) suspended in a liquid medium of the same composition were dropped, and these plates were incubated for 25 minutes.
The cells were statically cultured in the dark at ℃.

The protoplasts used in this experiment were obtained by treating the plant vesicles each time with cellulase and peptidase, and purifying the protoplasts by the density gradient method if necessary, and having a survival rate of 80% or more immediately after isolation.

Cultivate! After 3 days, these plates were observed under a microscope and 1
) Number of inoculated cells per plate, 2) Total number of dividing cells per plate were counted. From this value, division frequency [2) ÷ 1)
100] and calculated the average of 5 plates. The results are shown in Table 2. The division frequency of all plant cell protoplasts was higher in the supplemented medium.

Table 1 Composition of medium Table 2 Time 1 Wasabi L 48.8± 7.0 0,
5±0.31□ C: Callus, L: Leaf, R: Root, S: Stem, SC: Suspension cultured cell Example 2 The effect of adding albumin on cell density was investigated using tobacco mesophyll protoplasts as a test subject. .

Preparation of the medium, culture method, and examination of division frequency were performed according to Example 1. Division frequencies were determined by seeding 10, 100, and 1000 cells per plate. Here, ioo per plate.

If 100 protoplasts were inoculated, 100 cells were arbitrarily added to each plate. The frequency of division was calculated based on the results. The results are shown in Table 3. The 8 value is the average value obtained from 5 plates.

Table 3 Effect of cell concentration on division frequency Example 3 Using corn mesophyll protoplasts as the test subject,
The effects of albumin and globulin addition on cell division were investigated. All Sigma products were used in this experiment. Preparation of the medium, culture method, and examination of division frequency were performed according to Example 1. The results are shown in Table 4. The 8 value is the average value obtained from 5 plates.

These results show that various albumins are effective, but globulin is not.

Table 4 Albumin 1IA
7030 B Elaisa Certification Grade 62.3±10.1
1 albumin 1A7638 B 65.7± without globulin
6.81 Near 2. A4. ! 'EG C albumin-free 22.0±2.
81 No additives 19.3±3
．． 1°B: Bovine serum, C: Chicken egg white [Effects of the invention] Generally, in culturing plant cells, it is necessary to use a medium with a different composition depending on the individual cell type. However, according to the method of the present invention, even if any medium is used, the efficiency of cell division and proliferation can be greatly increased by adding a single substance, namely albumin. This will greatly encourage the development of plant cell culture technology.

Applicant's representative Patent attorney Takehiko Suzue Procedural amendment, B16 Nagisa Striker Director of the Patent Office Michibe Uga 1, Indication of case Patent Application No. 177206/1988 2, Title of invention Plant cell culture method 3, Make amendments Patent applicant (093) Gakken Co., Ltd. 4, agent Akira Mori Building, 1-26-5 Toranomon, Minato-ku, Tokyo, Japan
Particulars 61.2□26)
7. Contents of correction '+3.
−/Amend the claims as shown in the attached sheet.

``4''・吃'EN.

2. Claims (1) A method for culturing plant cells, which comprises culturing the plant cells in a medium supplemented with albumin.

(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.

(5) The plant cell culture method according to claim 1, wherein the albumin is bovine serum albumin.

(6) The plant cell culture method according to claim 1, wherein the albumin is chicken ovalbumin.

Claims (6)

[Claims] (1) A method for culturing plant cells, which comprises culturing the plant cells in a medium supplemented with 0.1% by weight of albumin. (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. (5) The plant cell culture method according to claim 1, wherein the albumin is bovine serum albumin. (6) The plant cell culture method according to claim 1, wherein the albumin is chicken ovalbumin.