JPH0763283B2 - Propagation of callus derived from saffron - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention is directed to iris saffron (Crocus sativus).
L.) for efficiently growing callus derived from the stigma.

[Conventional technology]

The stigma of pistil of saffron contains crocin, picrocrocin, safranal, etc. and is extremely important as a food coloring, fragrance and medicinal. Saffron is cultivated once a year, and the stigma that blooms is picked by hand, then dried to make it into a product. However, such a method requires a large cultivation area and manpower, is greatly affected by the weather, and the quality is not constant.

Utilization of plant tissue culture technology is considered as a means for solving these problems.

The stigma of saffron is cultured to induce callus, and the stigma-like tissue is redifferentiated from the cultured callus.
It was reported by Koyama et al. At the annual meeting. Mura is currently used in plant tissue culture.
shige & Skoog (hereinafter referred to as MS), Linsmaier & Sko
og (hereinafter referred to as LS), Nitsch & Nitsch (hereinafter referred to as NN), White (hereinafter referred to as WH), Gamborg, Miller &
Ojima (hereinafter referred to as B-5), Schenk & Hildebrand
There are media such as t (hereinafter referred to as SH) and Heller,
Among them, MS medium is generally used as a growth medium for callus derived from stigma.

This MS medium had a total nitrogen content of 840.8 mg /, and the ratio of ammonia nitrogen content to the total nitrogen content (hereinafter referred to as AN / TN) was 3
Although it is 4.3%, it is insufficient for efficiently growing callus derived from saffron stigma.

[Problems to be Solved by the Invention]

Conventional culture media have various problems, and in order to efficiently grow a large amount of stigma-derived saffron callus,
Not enough. The amount of total nitrogen and the ratio of the amount of ammonia nitrogen to the total amount of nitrogen were examined as a means for efficiently growing a large amount of this callus. In the present invention, the “total nitrogen amount” means the total weight of the nitrogen atom portion of the nitrogen-containing compound contained in the medium. Further, the “ammonia nitrogen amount” means the total weight of the nitrogen atom portion of the compound containing NH ₃ or NH ₄ ⁺ contained in the medium.

The present inventors examined the total nitrogen and ammonia nitrogen of the medium from the viewpoints of amount and ratio, and found a method for efficiently growing callus.

[Means for Solving the Problems]

The method for growing callus derived from the stigma of the saffron of the present invention has a total nitrogen content of explants derived from the stigma of saffron of 200 to 1000 mg /
And a callus growth medium having an AN / TN of 30% or less, preferably 3 to 15%, and the obtained callus is further grown in the medium. The present invention relates to a method for growing callus, and another step is required to redifferentiate the tissue from the grown callus.

The compound containing ammonia nitrogen used in the medium used in the present invention is not particularly limited, and examples thereof include ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium monohydrogen phosphate, ammonium dihydrogen phosphate, and ammonium carbonate. As the component, in addition to the compound containing ammonia nitrogen, a compound containing nitrate nitrogen, for example, potassium nitrate, sodium nitrate, calcium nitrate, magnesium nitrate, manganese nitrate, iron nitrate nonahydrate, zinc nitrate hexahydrate. A hydrate is mentioned.

The medium to be used in the present invention is a medium in which a normal inorganic component and a carbon source are essential components in addition to nitrogen, and phytohormones and vitamins can be added thereto if necessary. As inorganic components other than nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc,
Inorganic salts containing elements such as boron, molybdenum, chlorine, iodine and cobalt can be mentioned. Specific examples thereof include potassium nitrate, sodium nitrate, ammonium sulfate, calcium chloride, potassium monohydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate and magnesium chloride. , Sodium sulfate, ferrous sulfate, manganese sulfate, copper sulfate, sodium molybdate,
Examples thereof include compounds such as potassium iodide, zinc sulfate, boric acid and cobalt chloride.

Examples of the carbon source include carbohydrates such as sucrose and derivatives thereof.

Examples of the plant hormones include auxins such as 1-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid, and cytokinins such as benzyladenine (BA), kinetin, and zeatin.

Examples of the vitamins include thiamine, inositol, pyridoxine, nicotinic acid, folic acid and biotin.

Amino acids such as glycine, asparagine, glutamine, proline, serine and lysine may be added to the medium.

The medium of the present invention usually contains 0.1 μM to 90 m of inorganic components.
M, carbon source 10 g / to 90 g /, plant hormones 0.1
mg / to 100 mg /, vitamins 0.1 mg / to 100
It is preferable to use 0 mg / amino acid and 0 to 1000 mg / amino acid.

Examples of the medium used for the tissue culture of the present invention include those conventionally used for plant tissue culture, such as MS medium, LS medium, WH medium, B-5 medium, NN medium, and SH medium. Carbon and plant hormones are added, and if necessary, the above-mentioned vitamins and media are prepared by adding amino acids, but in the present invention, the total nitrogen content is particularly 400 mg / SH medium. A medium prepared by using the plant hormone concentration is preferably NAA 0.1 to 100 mg /, BA 0.1.
~ 20 mg / is preferred. Note that, regarding the composition of the conventionally known medium described above, for example, Yasuyuki Yamada "Plant Cell Culture Manual" p.6 to p.8, Kodansha Scientific,
Listed in 1984.

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 gellite.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but these do not limit the scope of the present invention.

Example 1 A treatment zone having a total culture nitrogen amount of 1/2 to 4.0 times 400 mg / (AN / TN 11%) was prepared, and plant hormone NAA 10 mg /, BA 1 mg /
The results of culturing for 42 days in the dark at 22 ° C are shown in Table 1. For components other than nitrogen, the culture components of SH medium are used, and sucrose is 3%.
And

Nitrogen concentration ratio 1 shows total nitrogen 400mg /, 1 / 2,2,4 is
It shows 200,800,1600 mg /, respectively. Nitrogen concentration ratio 1 / 2,2
The growth of callus was slightly inferior to that of 1, but the growth of 4 was about 1/2.

Comparative Example 1 The results of culturing in the medium of MS, SH, NN and WH under the same conditions as in Example 1 are shown in Table 2.

A ^* is the case where the nitrogen concentration ratio of Example 1 is 1.

The numbers in parentheses indicate the growth when the growth ratio of MS medium known as a growth medium for callus derived from saffron stigma is 100.

Cultures were performed using media having different total nitrogen contents and AN / TN.

From Table 2, it can be seen that the A ^* -treated group and the SH medium-treated group according to the present invention are obviously superior to the known MS medium-treated group in the growth efficiency.

Example 2 Table 3 shows a comparison of proliferation in Example 1 with the total nitrogen amount being 400 mg /, and the ratio of the amount of ammonia nitrogen (hereinafter referred to as AN) and the amount of nitrate nitrogen being varied. Other conditions are the same as those in the first embodiment.

The maximum growth rate was AN / TN 11.0%, and AN / TN 67.0% was 41, which was less than 1/2, and 83% at AN / TN 0%.
Met.

Example 3 Table 4 shows the growth amount and treatment group when AN was set to 44 mg / (AN / TN 11.0%) and nitrate nitrogen (hereinafter referred to as NN) (sodium nitrate was used) was further added. It was The plant hormone concentration, culture temperature, culture period, etc. are all the same as in Example 1. SH medium components were used for components other than nitrogen, and 3% was used for sucrose.

The growth ratio of callus gradually decreased with increasing NN addition amount, and when TN 1400 mg / was set, it markedly decreased,
The growth ratio was 41.

〔The invention's effect〕

According to the present invention, a large amount of callus derived from saffron stigma, which is a cultured tissue, can be efficiently proliferated.

Claims (1)

[Claims] 1. The total amount of nitrogen (weight of nitrogen atom portion) of the explant derived from saffron stigma is 200 to 1000 mg /, and the ratio of the amount of ammonia nitrogen (weight of nitrogen atom portion) to the total nitrogen amount. A method for proliferating callus derived from stigma of saffron, characterized in that the callus is proliferated using a callus proliferating medium having a ratio of 30% or less, and the obtained callus is further proliferated in the medium.