JP2952534B2 - Method of inducing oil and fat production callus - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for inducing coconut endosperm callus having a high oil-and-fat producing ability at a high rate.

[0002]

2. Description of the Related Art Fats and oils accumulated in coconut endosperm are mainly composed of medium-chain fatty acids and are important as raw materials for industrial and food fats and oils. However, the productivity of fats and oils from natural coconut fluctuates significantly depending on weather and environmental conditions. Therefore, by inducing and separating callus having high oil and fat production capacity, medium-chain fatty acids can be industrially mass-produced without being influenced by weather and environmental conditions. From this viewpoint, some attempts have been made to induce callus from coconut endosperm tissue. For example, Fi
Sher and Tsai have attempted callus induction using various endosperm tissues, but have only obtained one callus and reported that their growth was unstable [In Vitro.
Vol. 14, No. 3,307-311 (197
8)]. Also, P.I. Prakash Kumar et al. Describe that callus can be induced at a frequency of 30% or more from endosperm tissue contained in immature fruits, but the details of the conditions are unclear. Callus could not be induced well. Furthermore, it has been reported that the accumulation of fats and oils in calli disappears by repeating subculture [Plant Science]
e, 40 (1985), 203-207].

[0003]

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a method for stably inducing a high fat / oil-producing callus from coconut tissue at high frequency.

[0004]

Under such circumstances, the present inventors have studied the relationship between the degree of endosperm tissue development and the rate of callus induction,
Relationship between endosperm site and callus induction rate, callus oil and fat content,
As a result of various studies on the callus culture stability, etc., it was found that callus with a high oil-and-fat productivity can be induced with high frequency by culturing using the peripheral part of the embryo obtained from the fruit 6 to 9 months after pollination. Completed the invention.

[0005] That is, the present invention provides an oil / fat-producing callus characterized by culturing a peri-embryonic part of a coco endosperm tissue obtained from fruits harvested 6 to 9 months after pollination in a medium containing a plant growth regulator. Is provided.

Hereinafter, the present invention will be described in detail. In the endosperm tissue of coconut, cells are arranged in a film form along the endocarp a few months after pollination. Thereafter, the cell number increases due to cell division, and cell hypertrophy occurs. About 10 months after pollination, the endosperm tissue becomes hard, becomes a white layered tissue, and a large amount of fats and oils are accumulated in the cells. The endosperm tissue used in the present invention is obtained from fruits harvested 6 to 9 months after pollination. When the period exceeds 9 months after pollination, not only the tissue becomes firm, but also the induction of callus hardly occurs. The endosperm tissue 6 to 9 months after pollination has a tissue thickness of 2
It is a jelly-like or soft tissue that develops to 7 mm to 7 mm, and is easy to collect.

[0007] In the endosperm tissue, the peripheral part of the embryo has a specifically high callus induction rate, and the peripheral part of the embryo is used in the present invention.

[0008] The method of collecting the tissue around the embryo from the fruit is not particularly limited as long as it is sterile and does not kill the tissue.
After removing the mesocarp, sterilize the endocarp with ethanol, sodium hypochlorite, and hydrogen peroxide, make a hole in a germination hole in the endocarp, and collect endosperm tissue with a spoonful of medicine. . Here, endosperm tissue is extremely soft and should not be sterilized directly with a bactericide.

[0009] Next, the obtained peri-embryonic tissue is cultured in a medium containing a plant growth regulator. The medium used for such callus induction is Murashige and
Skoog (MS) medium, Gamburg's B-
5 medium, Euwens's Y-3 medium, White medium and the like. Examples of plant growth regulators to be added to the medium include auxins, cytokinins, gibberellins and the like. Auxins include 2,4-
Dichloroacetic acid (2,4-D), naphthaleneacetic acid (NA
A), indolebutyric acid (IBA), indoleacetic acid and the like, the concentration of which is 1 to 150 ppm, especially 20 to 1 ppm.
00 ppm is preferred. Examples of cytokinins include 6-benzyladenine (BA), zeatin, 2-isopentynyl adenine (2-iP), kinetin and the like.
The concentration is preferably 0.1 to 10 ppm. Gibberellin, abscisic acid and the like can also be added as other plant growth regulators. By further adding activated carbon to the medium, the browning of the tissue pieces can be prevented. Sucrose and glucose of 3 to 10% are suitable as saccharides, but other monosaccharides, oligosaccharides and polysaccharides can also be used.

Here, the culture is carried out at a temperature of 25 to 30 ° C., and the lighting conditions are not particularly limited.
It is performed for a period of up to two months, and this leads to a very high rate of callus induction.

The callus thus induced can accumulate a high concentration of fats and oils in the callus by subjecting it to growth culture. Propagation culture may be carried out, for example, using the medium used for callus induction described above, transplanted every month, and subcultured. In order to collect oils and fats from the callus after the growth and culture, a conventional method, for example, solvent extraction with petroleum ether, hexane or the like may be used. When the above Y-3 medium was used as the medium and the cells were grown and cultured for three months, the callus proliferative activity was about 40-fold. In addition, as a result of extracting the fat and oil from the callus subcultured for 20 months and examining the fatty acid composition,
It was revealed that lauric acid and myristic acid, which are features of coconut oil, are contained in large amounts. Furthermore, the presence of oil droplets in the cells was confirmed by histochemical techniques.

[0012]

Industrial Applicability According to the present invention, callus of coconut endosperm tissue having high oil and fat productivity can be induced at a high rate. By using the callus thus induced, oil and fat mainly containing medium-chain fatty acids can be industrially produced. It can be manufactured stably.

[0013]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Coconut fruit 5 to 10 months after pollination was harvested, the epicarp of the fruit was previously sterilized, the mesocarp was removed, and the inner pericarp was sterilized with ethanol. A hole was made in the germination hole in the endocarp, and the immature endosperm tissue was collected with a spoonful of medicine.
The tissue pieces were cultured in a Y-3 solid medium containing 20 ppm of 2,4-D, 1 ppm of BA, and 1 ppm of 2-iP plant growth regulator, 0.25% activated carbon, and 0.2% gellite. Table 1 shows the callus induction rate after two months. As a result, calli were induced from the endosperm tissue of the fruit from 6.0 months to 8.7 months after pollination, but no induction occurred at other fruit ages.

[0015]

[Table 1]

Example 2 In the same manner as in Example 1, endosperm tissues were collected from the fruit 7.3 months after pollination by site. The collection site is the peripheral part, the middle part, and the tip part (the part facing the embryo) of the embryo. Tissue pieces from each site were cultured, and the rates of callus induction from each site were compared. As shown in Table 2, it is clear that calli are induced at a high rate around the embryo.

[0017]

[Table 2]

Example 3 Endosperm tissues were collected from fruits 7.3 months after pollination in the same manner as in Example 1, and cultured in media containing various concentrations of auxin. Table 3 shows the results of the callus induction rate.

[0019]

[Table 3]

Example 4 The callus obtained in Example 2 was replaced with Y-3 described in Example 1.
Subculture was continued every 6 weeks on a solid medium, and about 500 mg of callus after 6 months were collected and oil was extracted with petroleum ether. To this, anhydrous methanol containing 1% sulfuric acid was added, and 9
Heated at 0 ° C. for 10 minutes. After cooling, the reaction solution was extracted again with petroleum ether, the aqueous phase was removed, sodium sulfate was added, the mixture was centrifuged at low speed, and the fatty acid methyl ester recovered in the supernatant was analyzed by gas chromatography. Identification of each fatty acid is determined by comparing the retention time with the standard,
The fatty acid composition of the oil was calculated from each peak area. The content varied depending on the callus strain, but was 10 to 60%, and was a fat containing lauric acid and myristic acid. Table 4 shows the fatty acid composition.

[0021]

[Table 4]

Continuation of the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 5/04 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A method for inducing oil-producing callus, comprising culturing an embryo-surrounding site of a coco endosperm tissue obtained from fruits harvested 6 to 9 months after pollination in a medium containing a plant growth regulator. .