JP2845481B2 - Culture method of Mishima psychoprotoplast - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for culturing Mishima saiko protoplasts, and the method of the present invention can be widely used for breeding Mishima saiko and producing saponins and the like.

[Prior art] In breeding of plants by plant tissue culture technology, techniques such as cell fusion and gene transfer are widely used, but in order to utilize these technologies, it is necessary to use a protoplast culture system in each plant species. Is essential.
Regarding the cultivation of protoplasts, since the tobacco was reported by Takebe et al. (Planta, 99 , 12-20, 1971), the cultivation of a number of plant species has been reported.
In recent years, there have been examples of successful cultivation of rice, which has been difficult to culture (Bio. Technology,
4 , 1085-1090, 1986; Plant CellReports, 5 , 85-8
8, 1986; Plant Science, 47 , 123-133, 1986). However, different plant species have different culturing methods and culturing conditions, and successful culturing methods for other species cannot always be applied. It is necessary to establish a protoplast culture system for each plant species.

Mishima saiko is an important medicinal plant that has been widely used as a crude drug as an anti-inflammatory, antipyretic, or analgesic, but there has been no example of successful protoplast culture.

[Problems to be solved by the invention]

Therefore, in order to breed an important medicinal plant, mishima saiko, using a plant tissue culture technique, it has been desired to develop a protoplast culture system suitable for the mishima saiko protoplast. The present inventors isolated a protoplast derived from Mishima Saiko according to a conventional method, purified it, and then cultivated it under various conditions, and examined the culturing conditions, and found that plant tissues containing specific vitamins or organic acids, etc. It was found that when protoplasts were cultured in a culture medium, the protoplasts started dividing, and eventually formed calli.

[Means for solving the problem]

The present invention, nicotinic acid, pyridoxine hydrochloride, thiamine hydrochloride, calcium D- pantothenate, p- aminobenzoic acid, choline chloride, ascorbic acid, vitamin A, vitamin D _3, vitamin B _12, sodium pyruvate, citric acid, malic The present invention relates to a method for culturing a protoplast derived from Mishimasaiko (Bupleureum), which comprises culturing in a plant tissue culture medium containing sodium acid, fumaric acid, and casamino acid.

 Hereinafter, the present invention will be described in more detail.

Protoplasts are obtained by slowly immersing the cells of the mishima saiko in an enzyme solution containing cellulase and pectinase, the osmotic pressure of which is adjusted with mannitol, for 2 to 3 hours in accordance with a conventional method. After washing the obtained protoplasts with a washing solution, it can be used for culture.

The culture medium for protoplast culture used in the method of the present invention is nicotinic acid 0.1 to 5.0 μg / ml (preferably 0.5 to 1.0 μg / m
l), pyridoxine hydrochloride 0.1-5.0 μg / ml (preferably 0.5
1.01.0 μg / ml), thiamine hydrochloride 0.1 to 20.0 μg / ml (preferably 1.0 to 10.0 μg / ml), calcium D-pantothenate
0.1-5.0 μg / ml (preferably 0.5-1.0 μg / ml), p-aminobenzoic acid 0.01-2.0 μg / ml (preferably 0.01-0.5 μg
g / ml), choline chloride 0.1-5.0 μg / ml (preferably 0.5-
1.0 μg / ml), ascorbic acid 0.1-10.0 μg / ml (preferably 0.1-5.0 μg / ml), vitamin A 0.01-1.0 μg / ml (preferably 0.01-0.1 μg / ml), vitamin D ₃ 0.01- 1.0μg /
ml (preferably 0.01~0.1μg / ml), vitamin B ₁₂ 0.01 to
1.0 μg / ml (preferably 0.01 to 0.1 μg / ml), sodium pyruvate 1.0 to 40.0 μg / ml (preferably 10.0 to 30.0 μg
/ ml), citric acid 10.0-80.0 μg / ml (preferably 20.0-5
0.0μg / ml), sodium malate 10.0 ~ 80.0μg / ml
(Preferably 20.0-50.0 μg / ml), fumaric acid 10.0-80.0
μg / ml (preferably 20.0 to 50.0 μg / ml) and / or casamino acid 50.0 to 500.0 μg / ml (preferably 100.0 to 300.0 μg
/ ml). The protoplast culture medium used in the method of the present invention contains, in addition to the essential components such as vitamins and organic acids, auxins as plant hormones in an amount of 0.5%.
５5 μg / ml, preferably 1.0-2.0 μg / ml, and one carbon source
-10%, preferably 3-6%, and the osmotic pressure is preferably adjusted to 0.3M-0.7M, preferably 0.4M-0.5M with mannitol. As auxins,
2,4-dichlorophenoxyacetic acid (hereinafter abbreviated as 2,4-D),
α-naphthaleneacetic acid (hereinafter abbreviated as NAA), indole-
3-acetic acid, indole-3-butyric acid, 2,4,5-trichlorophenoxyacetic acid and the like can be added. Glucose or sucrose can be used as the carbon source. Preferably, the osmotic pressure is adjusted with glucose or sucrose deficient with metabolically inert sugars such as mannitol or sorbitol. The basic medium of the plant tissue culture medium used in the present invention includes Murashige & Skoog medium, rinse car and Skoog (Linsmaicr & Skoog) medium (hereinafter abbreviated as LS medium), V47 medium, V-KM medium , B5 medium, KM8p medium or WB
A culture medium can be used, and a culture medium composed of only the inorganic salts of the above-mentioned culture medium may be used.

Since protoplasts derived from mishimasaiko have strong cohesiveness, if they are dispersed in a liquid medium as they are, they will soon aggregate and are not preferable for subsequent division. Therefore, it is desirable to use a technique of embedding protoplasts in a medium with a coagulant. The protoplasts embedded in the medium as described above are cultured at about 20 ° C. to 30 ° C. in the dark or under illumination. Colonies form after about 30 to 40 days. The colonies thus obtained can be transplanted to an appropriate medium by a conventional method to form callus. By acclimating and growing the obtained callus by a conventional method, a mishimasaiko plant can be obtained.

〔Example〕

Next, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following embodiments.

A callus derived from the leaves, stems, or roots of Shishima saiko (Bupleurum falcatum L.) according to a conventional method is suspended in an LS liquid medium supplemented with 0.5 μg / ml of 2,4-D as a plant hormone, and liquid culture is performed. Was. The liquid suspension cultured cells were subcultured every week, and those with vigorous growth were used as the material for protoplast isolation.

An enzyme solution containing cellulase and pectinase, whose osmotic pressure was adjusted to 0.5 M with mannitol, was added to the liquid suspension cultured cells, and the cells were slowly immersed at 27 ° C. in a dark place. After performing the enzyme treatment for about 2.5 hours, the mixture was filtered through an 82 μm nylon mesh to remove untreated cell mass and cell debris. The obtained protoplasts were washed with a washing solution having the following composition.

_{KH 2 PO 4 27.2 mg / KNO} 3 101 mg / CaCl 2 · 2H 2 O 1480 mg / MgSO 4 240 mg / KI 0.16 mg / CuSO 4 · 5H 2 O 0.025mg / mannitol 90 g / Meanwhile, shown in Table 1 Basic mediums 1 to 4 each having a composition are prepared, and a plant hormone and a carbon source are added to each of the basic mediums 1 to 4 as shown in Table 2 and the osmotic pressure is adjusted with mannitol to obtain a medium for plant tissue culture. Was prepared.

Washed protoplasts were embedded in each of the thus obtained culture media at a concentration of 2 × 10 ^{4 to} 5 × 10 ⁵ cells / ml (see Table 2), and culture was started at 27 ° C. in a dark place. In addition, 0.15% of gellan gum was used as a coagulant. Protoplasts start the first division 2-3 days after the start of culture, and after 1-2 months
It grew to a colony of about 0.2 to 0.5 mm. Table 2 shows the results of the frequency of appearance of colonies. The colonies that appeared in this manner were transformed into a normal callus subculture medium (2,4-D 1
When transplanted to LS agar medium supplemented with 0.1 ppm of KT and KT, the callus grew smoothly to calli.

──────────────────────────────────────────────────続 き Continued on the front page (56) References Physilogia Plant arum, Vol. 39, No. 4, p. 257-260 (1977) Physilogia Plant arum, Vol. 72, No. 2, p. 337-342 (1988) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 5/04 CA (STN)

Claims (1)

(57) [Claims] 1. A nicotinic acid, pyridoxine hydrochloride, thiamine hydrochloride, calcium D- pantothenate, p- aminobenzoic acid, choline chloride, ascorbic acid, vitamin A, vitamin D _3, vitamin B _12, sodium pyruvate, citric acid, A method for culturing a protoplast derived from Mishimasaiko (Bupleurum), comprising culturing in a plant tissue culture medium containing sodium malate, fumaric acid, and casamino acid.