JP2502308B2 - Method for culturing protoplasts of herbaceous plant - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to culturing plant protoplasts by a specific method to cause large and rapid division of protoplasts to proliferate cell clusters.

[Conventional technology]

In recent years, with the development of tissue culture technology, it has become possible to proliferate plant protoplasts into callus cells, regenerate the plant body, and grow new hybrid plants by cell fusion. Technically, plant regeneration and production of new species plants are performed through the following operations.

(1) Protoplasts are isolated from plant tissues or callus cells.

(2) Fusion of different types of protoplasts.

(3) Proliferate cell clusters from protoplasts or fused protoplasts.

(4) Regenerate the plant from the cell mass.

Despite the fact that it is expected to bring extremely high value industrially, there are actually many problems with this technology. In particular, the technologies other than the above (1) are problems that need to be solved. The present applicants have previously shown a solution to the above (2) and (4) in Japanese Patent Application Laid-Open No. 59-216589 or Japanese Patent Application No. 61-308539. On the other hand, regarding (3), attempts have been made to increase the division efficiency of protoplasts by examining the type of medium and the type and concentration of plant hormones, but no effective solution has been found. Recently, ornithine in protoplast cultures of alder and almond,
It has been reported that division is observed by the addition of arginine amino acid and putrescine, which is a kind of polyamine. (Plant Science Letters, vol 28,3-9,1982 and Plant Science, vol 41,55-60 1985) In the method,
Amino acids and polyamines are used alone. However, whether or not such amino acids and polyamines are effective as mitogens in protoplast culture of herbaceous plants has not been examined, and it is expected to establish a more effective method.

[Problems to be solved by the invention]

The present inventors have recognized that the conventional method for culturing protoplasts has various problems, and the method for culturing protoplasts by a novel method different from the conventional method to efficiently obtain a cell mass. Was examined.

[Outline of Invention]

As a result, the present inventors have discovered a substance that acts on plant protoplasts to promote cell division, and based on these findings, discovered a method for efficiently proliferating cell clusters from protoplasts. That is, according to the method of the present invention,
A method for culturing a protoplast of a herbaceous plant is provided, which comprises using a medium containing a polyamine in isolating and culturing the protoplast from a tissue or a cultured cell of the herbaceous plant.

[Specific Description of the Invention]

The plants to which the protoplast culture method according to the present invention can be applied are herbaceous plants. Specific examples of the plant include purple, Japanese cabbage, Japanese radish, petunia, tomato, Chinese cabbage, Hiroshima mana, bok choy, tobacco, lily, cucumber, rice and the like.

In the present invention, protoplasts can be isolated from the tissues or cultured cells of the plant and used. Specific examples of the tissue include hypocotyl, cotyledon, leaf, stem, and other tissues. These tissues are usually used after being sterilized with sodium hypochlorite or ethyl alcohol. However, when using aseptically cultivated plants, the above sterilization operation is unnecessary. When a disease-free and virus-free plant is used, the tissue near the growth point, the above-described tissue of the plant obtained from the tissue near the growth point, or the like can be used as the culture material. The cultured cells that can be used in the tissue culture of the plant of the present invention are undifferentiated atypical cells including callus tissue obtained by culturing the tissue piece by a known method.

In the present invention, the following method is used for culturing plant protoplasts to obtain a cell mass.

In the present invention, protoplasts can be isolated from tissues or cultured cells of the plant using enzymes such as macerozyme and cellulase. Regarding the enzyme concentration, processing time, etc.,
For example, "Introduction to Plant Cell Breeding" by Hirai, Naiku, and Sugiura, P2
1 to P38, Academic Publishing Center, 1982.

In the present invention, at least one selected from polyamines is used as a method for promoting the growth of isolated protoplasts.
A method of culturing using a medium to which seeds, preferably three kinds of compounds are added is used. At that time, addition of at least one amino acid such as glutamine, proline, and arginine to the medium in addition to polyamine further promotes the growth of protoplasts. Therefore, addition of amino acid is desirable. According to this method, the protoplasts are significantly promoted to divide and proliferate into cell clusters. It is a novel finding of the present inventor that division is promoted when protoplast culture of a herbaceous plant is carried out using a medium to which such a specific compound is added.

The medium that can be used in the present invention is a medium in which an inorganic component and a carbon source are essential components, and plant hormones and vitamins are added thereto. The inorganic components of the medium include nitrogen, phosphorus, potassium, sodium, calcium,
Magnesium, sulfur, iron, manganese, zinc, boron,
Inorganic salts containing elements such as molybdenum, chlorine, iodine and cobalt can be mentioned, and specifically, potassium nitrate, ammonium nitrate, calcium chloride, potassium monohydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, magnesium chloride, sulfuric acid. Sodium, ferrous sulfate, ferric sulfate
Iron, manganese sulfate, copper sulfate, sodium molybdate,
Examples thereof include compounds such as molybdenum trioxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride.

Examples of the carbon source of the medium include carbohydrates such as sucrose and derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol.

Examples of the plant hormones in the medium include naphthalene acetic acid (NAA), indole acetic acid (IAA), p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D),
Auxins such as indole butyric acid (IBA) and their derivatives, and benzyladenine (BA), kinetin,
Cytokinins such as zeatin can be exemplified.

The vitamins in the medium include biotin, thiamine (vitamin B ₁ ), pyridoxine (vitamin B ₆ ), pyridoxal, pyridoxamine, calcium pantothenate,
Ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide and riboflavin (vitamin
B ₂ ).

Examples of the osmotic pressure regulator of the medium include carbohydrates such as mannitol and compounds such as potassium chloride.

The medium of the present invention usually contains the inorganic component in an amount of about 0.1.
μM to about 100 mM, about 10 g / l to about 100 g of the carbon source
/ l, about 0.01 mg / l to about 10 mg / l of the plant hormones,
It is preferable to use the vitamins in an amount of about 0.1 mg / l to about 150 mg / l and the osmotic pressure adjusting agent in an amount of about 0.2M to about 1M.

Specific examples of the medium used for tissue culture according to the present invention include mediums conventionally used for tissue culture of plants, for example, Murashige Skoog ('6
2) [Murashige & Skoog] Medium, Rinse Meyer
Skug (RM-1965) [Linsmaier & Skoog] medium,
White ('63) [White] medium, Gambog
rg) B-5 medium, Mitsui M-9 medium, Nitchi / Nitch medium [Nitch & Nitch] Kao et al. 8P and A.
Add the above-mentioned carbon source and plant hormones to the medium,
Further, a medium prepared by adding the above-mentioned vitamins as required can be exemplified.
A medium prepared using -5, 8P or A medium is preferred. Incidentally, regarding the composition of the conventionally known medium described above,
For example, Harada and Komine, "Plant Cell Tissue Culture" P390-P39
1, Rikagakusha, 1979, Planta, vol 126, 105-110, 1975 or In Vitro, vol 17, 645-648, 1981.

The medium that can be used in the present invention is a liquid medium or a solid medium containing 0.2 to 1% of agar or agarose.

In the present invention, the concentration of the polyamine added to the above-mentioned medium in the medium is usually 10 ⁻⁸ to 10 ⁻⁴ M / l, preferably 10 ^−7.
It is in the range of ~ 10 ^-5 M / l.

Here, the polyamine added to the medium in the present invention is a compound having a structural unit having an amino group and / or an imino group at both ends of a polymethylene group [-(CH ₂ ) _n- , n is an integer] Is spermine [Bis (aminopropy
l) -tetramethylenediamine; H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (C
H _{2) 3} NH _2], spermidine _{[H 2 N (CH 2) 3} NH (CH 2) 4 NH 2 ] and putrescine _{[H 2 N (CH 2) 4} NH 2 ] can be exemplified tetramethylenediamine such as .

Examples of the amino acid to be added together with the polyamine include glutamine, proline, arginine, serine, alanine and the like, and the concentration thereof is usually 10 ⁻⁸ to 10 ⁻⁴ M / l, preferably 10 ⁻⁷ to 10 ⁻⁵ M / l. Is in the range.

According to the method of the present invention, a large amount of cell mass can be efficiently obtained from protoplasts of a herbaceous plant. To further refer to this point, the present invention can be applied to the growth of cell clusters from hybrid protoplasts obtained by cell fusion, and Japanese Patent Application No.
Protoplast-derived plants can be easily grown by using seedling growth methods such as -308539, 61-309540, and a method of injecting calmogyurin or carmosium into cells and then culturing.

〔The invention's effect〕

When the method for culturing a protoplast of a herbaceous plant according to the present invention is used, cell division can be promoted from the protoplast more efficiently than in the conventional method, and thereby a large amount of cell clusters can be proliferated.

〔Example〕

Hereinafter, the configuration and effects of the present invention will be specifically described with reference to examples.

Example 1 After isolating protoplasts from the hypocotyl of Hiroshima mana sterilized by raising seeds by a known method, sorbitol 4.6%, mannitol 4.6%, glucose 0.25%, naphthalene acetic acid 10 ⁻⁷ M, 2,4 -Dichlorophenoxyacetic acid 3 × 10
^-6 M, benzyladenine 10 ^-7 M, amino acids glutamine, proline, arginine, serine and alanine, respectively
As a polyamine according to the present invention, 10 ⁻⁶ M and putrescine, spermine, and spermidine were respectively contained at a concentration of 10 ⁻⁶ M, and a sterile Kha's A medium having a pH of 5.7 was prepared, and the protoplast of Hiroshimana was added thereto. When added and cultured for 10 days in the dark at 25 ° C, the results shown in Table 1 as the division rate were obtained. In addition, when the cells were cultured for 2 months, the results shown in Table 1 were obtained as the formation rate of cell clusters divided into 100 cells or more. In each of the treated samples, the division rate and the cell mass formation rate were both Comparative Example 1.
Increased compared to ~ 2.

Comparative Example 1 Table 1 shows the results of culturing Hiroshima protoplasts in the same manner as in Example 1 except that the medium did not contain the polyamine and the amino acid according to the present invention.

Comparative Example 2 Table 1 shows the results of culturing Hiroshima mana protoplasts in the same manner as in Example 1 except that the medium does not contain the polyamine according to the present invention.

Example 2 Table 1 shows the results of culturing Hiroshima mana protoplasts in the same manner as in Example 1 except that the medium did not contain amino acids.

Example 3 Mannitol, after isolation of protoplasts by known methods from sterilized seed-grown pettiunia leaves
6.4%, sucrose 2%, 2,4-dichlorophenoxyacetic acid 6 × 1
PH- ⁶ containing 0 ^-6 M, benzyladenine 2 × 10 ^-6 M, and polyamine according to the present invention containing putrescine, spermine, spermidine, amino acids glutamine, alanine, serine, proline and arginine at a concentration of 10 ^-6 M each. The sterile DPD medium of Example 7 was prepared, the protoplasts of petunia were added thereto, and the mixture was cultured at 25 ° C. for 14 days in the dark, and the results shown in Table 1 as the mitotic rate were obtained. When cultured for 2 months, the results shown in Table 1 were obtained as the formation rate of cell clusters divided into 100 cells or more. In each of the treated samples, both the division rate and the cell mass formation rate increased as compared with Comparative Examples 3 and 4.

Comparative Example 3 Table 1 shows the results of culturing protoplasts of pettiunia in the same manner as in Example 3 except that the medium did not contain the polyamine and amino acid according to the present invention.

Comparative Example 4 The results of culturing protoplasts of petunia in the same manner as in Example 3 except that the medium does not contain polyamine are shown in Table 1.

Example 4 Table 1 shows the results of culturing protoplasts of pettiunia in the same manner as in Example 3 except that the medium did not contain an amino acid.

Examples 5 to 14 In Example 3, radish hypocotyl, cabbage hypocotyl, tomato leaf, Chinese cabbage seedling, tobacco leaf, cucumber leaf, gun lily leaf, purple cultivated cell, rice cultivated cell, and bok choy leaf are used in Example 3. Table 2 shows the results of culturing protoplasts in the same manner as in the Example except for the above.

Comparative Examples 5-14 In Examples 5-14, radish, cabbage, tomato, Chinese cabbage, tobacco, cucumber, gun lily, gun lily, purple melon, in the same manner as in Examples 5-14 except that the medium does not contain the polyamine and amino acid according to the present invention, The results of culturing the protoplasts of rice and bok choy are shown in Table 2.

Claims (4)

(57) [Claims] 1. A method for culturing a protoplast of a herbaceous plant, which comprises using a medium containing a polyamine in isolating and culturing the protoplast from a tissue or a cultured cell of the herbaceous plant. 2. The method according to claim 1, wherein the medium is a medium containing polyamine and amino acid. 3. The method according to claim (1) or (2), wherein the polyamine is spermine, spermidine and putrescine. 4. The method according to claim 1 or 2, wherein the amino acids are glutamine, proline, arginine, serine and alanine.