JP2795542B2 - Method for producing taxol and taxanes from embryo culture of yew species - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method of producing taxol and its derivatives from somatic embryos and / or culture media,
Specifically, zygote embryos or somatic embryos are cultured to produce somatic poultry embryos (somatic po
ly-embryo) or embryogenic calli (embryogenic calli)
On how to get.

2. Description of the Prior Art Taxol is an alkaloid isolated from plants belonging to the genus Taxus and exhibits useful antitumor activity in various cancer cell lines including B16 melanoma.
Taxol is primarily tax brevifolia (Taxus
brevifolia) on the bark, approximately 0.
It is known to contain 02%.

Although the total synthesis of taxol has been successful,
This method is not considered suitable for commercial scale production of taxol due to its high cost. In addition, the semi-synthesis of taxol-like compounds (eg, taxorete) has also been successful using baccatin III or 10-deacetylbaccatin III. However, this approach faces exactly the same problems as described above.

Therefore, taxol can only be obtained from scarce natural resources such as, for example, the raw material of felled yew. Taxol content in yew trees is very low, so 2000-400 to collect 1 kg of taxol
Zero trees must be cut out and producing taxol from natural resources to meet demand will promote significant destruction of forest ecosystems. Accordingly, attempts have been made to produce taxol and related compounds from cell and tissue cultures of Taxus species.

For example, U.S. Pat.No. 5,019,504 teaches cultivating bark, cambium, leaf and root tissues of a yew species in a nutrient medium to produce callus or suspension cell cultures, and then recovering taxol or taxol-like alkaloids therefrom. A method for producing taxol or a taxol-like alkaloid is disclosed. However, the method described above requires only 1.0 liter of cell suspension culture medium.
It can only produce ~ 3.0 mg of taxol. This patent teaches that the yew tree resin can directly produce optimal yields of taxol, and by culturing these tissues to induce undifferentiated cell mass (callus), and Taxol is produced by establishing a cell suspension culture from the grown calli.

Still, the methods using various parts of the yew tissue are inadequate for the techniques developed for the low taxol-producing ability of the cells involved or for the large-scale production of secondary metabolites of interest. Because of this, it is not offered to the market.

WO92-13961 discloses a method for producing taxol by culturing a tissue of a plant belonging to the genus Taxus, particularly a female gametophyte, and recovering taxol from potash or suspension cultured cells derived from the tissue. I do.
This technique can provide 0.05% taxol based on the dry weight of the suspension culture cells.

These methods do not produce sufficient amounts of taxol on an industrial scale and are currently under development to improve taxol production. These facts indicate that cells and / or
Or to increase the taxol content in the culture medium,
As well as suggesting the need to provide improved methods for screening high taxol producing cells to meet the end purpose of industrial scale production.

In view of these circumstances, the present inventors have conducted intensive studies to develop a method for producing taxol on an industrial scale, and as a result, we have first, surprisingly, We found that zygotic embryos contained higher amounts of taxol on a dry weight basis than the other tissues reported.

Because the size of the embryo itself is too small to develop an industrial-scale approach to produce taxol, the inventors have conducted further studies to provide a method for increasing embryo volume. As a result, we have surprisingly found that embryos can be hundreds of thousands of times larger than their original size when cultured in a nutrient medium that induces somatic embryos or embryogenic calli.

 The present invention is accomplished based on the above findings.

SUMMARY OF THE INVENTION As described above, an object of the present invention is to provide a method for producing taxol by extracting a tissue of a yew species, wherein the tissue is a zygotic embryo.

It is another object of the present invention to provide a method for producing taxol or a derivative thereof by culturing a tissue of a yew species, and recovering taxol or a derivative thereof from callus or a culture medium. It is a somatic embryo.

Yet another object of the present invention is to provide a method for producing taxol or a derivative thereof, comprising the following steps: (a) preparing a zygote embryo from seed of yew species and disinfecting it (B) culturing the disinfected embryo in a culture medium to produce callus from the embryo; (c) culturing the callus obtained in (b) to produce a somatic embryo from the callus; (D) culturing the disinfected embryo in (a) or the somatic embryo obtained in (c) to produce embryogenic callus; (e) the somatic embryo in (c) or the embryo in (d) (B) recovering taxol or a taxol derivative from the culture medium and cells.

The present invention is based on the finding that zygotic embryos contain a surprisingly large amount of taxol and further increase embryo volume by hundreds of thousands through induction of somatic embryos or embryogenic callus. The present invention is distinguished from the prior art referred to above in that it makes it possible to produce taxol on an industrial scale by liquid culture of somatic embryos or embryogenic calli. Beneficial than prior art.

Other features and applications of the present invention will be readily apparent to one skilled in the art from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 (A) is an HPLC chromatograph of standard taxanes.

FIG. 1 (B) is an HPLC chromatograph of the purified culture medium obtained from Example 7.

FIG. 2 (A) is a photograph showing rat cancer cells without treatment in Experimental Example 1.

FIG. 2 (B) is a photograph showing rat cancer cells in Experimental Example 1 treated with the extract of Example 7.

 FIG. 3A is a standard curve based on ELISA data.

FIG. 3B is a curve based on ELISA data performed in Experimental Example 2.

FIG. 4 is a graph showing the effect of the production medium on taxol production in Example 8.

DETAILED DESCRIPTION OF THE INVENTION We have been planted in a national forest
Taxol content of 4,000 Taxus cuspidata trees was analyzed. The amount varied significantly between individual trees, but it was found that 5 g of taxol was contained in 100 g of dried embryos collected from the superior trees. On the other hand, the same amount of dried bark and leaves yielded 4.5 mg and 2.0 mg of taxol, respectively.

Therefore, a first object of the present invention is to provide a method for producing taxol, wherein taxol is extracted from the yew species embryo. The type of the yew species is not limited, and any tree can be used as long as it belongs to the genus Yew. Again, Taxus cuspidata is advantageously used.

The extraction method for producing taxol from the embryo is not particularly limited. However, in general, the extraction of taxol from embryos can be performed by immersing the lyophilized powder of the embryo in alcohol, especially methanol.

Unfortunately, zygote embryos are very small in size and embryos contain more than 90% water. Huge amounts of embryos are required to produce very small amounts of taxol, and extracting taxol directly from embryos is disadvantageous from an economic point of view.

Accordingly, the present inventors have intensively studied to provide a method for producing taxol economically without the above-mentioned problems, and as a result, by using tissue culture technology, especially somatic embryos and embryogenic callus The amount of embryos could be increased 100,000 times through the induction of E. coli. Therefore, a second object of the present invention is to derive a somatic embryo or embryogenic callus from a zygote of a Taxus species and to use the somatic cell or embryogenic callus in a liquid medium by using a shaker or bioreactor. It is to provide a method for producing taxol or a taxol derivative by culturing and recovering taxol from the culture medium and the cells.

One feature of the present invention is that the present invention can increase embryo volume from 10,000 to 100,000-fold through the induction of somatic embryos or embryogenic callus, thus presumably cell division of the meristem used. It is difficult for simple callus induction from various tissues other than embryos to reach such amplifications due to their different capacities. In particular, the induction of embryogenic calli makes it possible to increase the amount of embryos by a factor of 100,000 and is therefore very important.

The induction of somatic or embryogenic callus from zygotic embryos and the production of taxol or its derivatives are described below.

Mature or immature seeds collected from yew species during the period from August to November to aseptically culture embryos should be disinfected or surface sterilized. Disinfection or surface sterilization is performed using conventional techniques.
For example, seeds are soaked in 70% ethanol for 30-60 seconds, washed two or three times with sterile water, and surface sterilized with a 1-3% (v / v) solution of sodium hypochlorite for 24 hours.
The surface-sterilized seeds are rinsed with sterile water at least 5 times to separate the embryos.

Embryos may be used to extract taxol by following the extraction techniques described above.

Embryos are also placed on solid nutrient media to induce callus therefrom. Induction of somatic embryos can be performed using PEDC (Pre-Embryogenic Determined Cell) or IEDC (Induced Embryogenic Determined Cell).
Determined Cell)) method.

The induction of somatic embryos using the PEDC pattern is as follows.

1-naphthaleneacetic acid (hereinafter referred to as “NA
A "), embryos are cultured on solid nutrient media supplemented with kinetin and 2,4-D. Once callus has been induced, small pieces of potassium may be grown by culturing on media containing appropriate concentrations of macronutrients, micronutrients, and vitamins.

For the purpose of callus induction and growth, customary plant tissue culture media can be used without limitation. For example, mB
₅ (variant Gamborg B ₅ medium (modified Gamborg's B ₅ med
ium)), Durzan, MS (Murashihge & Skoog mediu
m), WPM (Lloyd & McCown), DKW (Driver-Kuniyuk)
-Walnut), GD (Gresshoff & Doy), SH (Schenk &
Hildebrandt medium) or LP (Quoirin & Lepiove)
r) may be used.

It is understood that these media may be modified such as by adding or deleting various components or changing the proportions. Among other media, mB ₅ or Durzan medium is preferred. As an intermediate step for somatic embryo development, a medium for induction that is the same as or different from the medium for rapid growth of callus may be used. From the standpoint of being able to obtain a maximum number of somatic embryos, mB ₅ medium is advantageously used.

Most of the above-mentioned media are commercially available or widely used, so that anyone can easily obtain it.
Therefore, the determination and / or optimization of a suitable culture medium for callus induction and rapid growth is within the ability of those skilled in the art. For example, mB ₅ medium to be advantageously used in the present invention is a medium slightly change the Gamborg's B ₅ medium, shows the composition shown in Table 1.

Furthermore, the IEDC pattern may be performed by culturing the embryo in the above-mentioned medium to which a different plant growth hormone has been added alternatively for the production of a somatic embryo.

For initial cultures, 2-4 ppm of 2,4-D is added. Callus is formed from the surface of the embryo and the cell volume is 10 ~
With a 20-fold increase, subculturing was usually continued by using the same medium with half the concentration of the auxin used.

Most calli lose their growth potential, presumably due to phenolic compounds secreted from non-proliferating cells, so supplemented with 0.5% (w / v) activated carbon or 1-2% (w / v) polyvinylpyrrolidine . The callus is subcultured for 2-4 months on a growth regulator-free medium to form somatic embryos from callus meristems.

The somatic embryo thus formed contains a large amount of taxol or a taxol derivative (hereinafter, “taxanes”) and can be used as such to extract taxanes. The extraction of taxanes from somatic embryos may be performed using a conventional method, particularly the method described below. However, somatic embryos are advantageously used to induce embryogenic callus to further increase cell volume.

According to the present invention, a relatively high rate of taxanes can be obtained by extracting not only somatic embryos produced as described above but also embryogenic potash. PEDC mentioned above or
By culturing somatic embryos or zygote embryos obtained by the IEDC method, embryogenic callus can be produced. The method of producing embryogenic callus is as follows. 1.0 to 4.0 ppm N
AA and 0.5-2.0 ppm kinetin, preferably 2.0 ppm
The disinfected or somatic embryos are cultured for 2-4 months in a solid medium suitable for inducing callus, supplemented with NAA and 1.0 ppm kinetin. 1-4 ppm of 2,4-D may be added to the medium instead of NAA plasticine. The induced callus is then cultured for 1 to 2 months in the same medium supplemented with 1 to 5 ppm of cytokinin such as benzylamine purine, N-isopentenylaminopurine, kinetin or zeatin, and embryo-like on the callus surface. The embryoid bodies were allowed to form and embryoid bodies were grown in the same medium supplemented with 2-10 ppm of 2,4-D at about 26 ° C. for 2-3 months, 16/8 hours (light / light).
Cultured under (dark) conditions. This culture makes it possible to obtain embryogenic callus in a large amount of 100,000 times the amount of the original embryo. Embryonic callus, for example, from yellow, brown to green,
It showed a wide range of colors.

For induction and growth of embryogenic calli, any medium that can be used for common plant cell and tissue culture may be used without limitation.

Embryogenic calli produced as described above contain large amounts of taxol and can be used directly for the extraction of taxol and taxanes.

Extraction of embryogenic callus may be performed by conventional techniques, in particular by the methods described below.

To amplify the amount of embryogenic callus on an industrial scale,
Embryogenic calli are aseptically sliced to produce single cell or small cell clumps. To establish a cell suspension culture, 10% (v / v) of these cells were seeded in a 250 ml conical flask containing 50 ml of liquid culture medium supplemented with 2 ppm of 2,4-D. An air-lift or impeller type bioreactor is advantageously used for this purpose in terms of continuous culture.

Bioreactor culture can be performed using two different culture media: one for cell growth (growth media) and another for the production of taxanes (production media). Growth medium preferably mB ₅ medium supplemented with 2,4 of 2～4Ppm, the production medium is preferably MS medium supplemented with NAA of 2 to 10 ppm.

In particular, the production medium preferably contains an elicitor to increase taxol production. The elicitor is a fungal elicitor, a female gametophyte extract (1-5 ml /
l), phenylalanine (50-300 mM) and G3A (0.5-
1.0 ppm). Examples of fungal elicitors
Cytospora abietis ATCC No. 38688 and Penicillium m
inioluteum (Dierckx), NRRL 18467, which are ground and subjected to a series of extractions to give an extract. Alternatively, it is also preferable to collect cultures of the genus pestalotiopsis and add the extract to the culture medium in an amount of 0.5 to 1.0 ml / l.

Taxol is not only present intracellularly, but is also secreted into the culture medium. Therefore, after the liquid culture process,
Taxols and taxanes can be recovered from the culture or culture medium using well-known techniques. Separation of cells from the culture medium is accomplished by centrifugation (eg,
g, 5-10 minutes) or by decantation. If the decantation technique is used for separation, the culture broth is allowed to settle for 24 hours to settle the cells, and the culture medium is decanted. The culture medium still remaining in the collected cells is removed by vacuum suction using a Pasteur pipette.

Taxanes could be recovered from somatic embryos or embryogenic callus cells as well as culture media by extraction with alcohol or a 1: 1 mixture of methanol and methylene chloride. The precipitate formed on centrifugation may be ground using a sonicator (Branson 250) and subjected to extraction.

According to the present invention, not only taxol itself, but also various taxol derivatives are recovered. Taxol derivatives include 10-deacetylbaccatin III, baccatin III, 10
-Deacetyltaxol, cephanormanine and 7-
Epi-10-deacetyltaxol is included (FIG. 1).
(See (B)). The taxol derivative may be used as it is or, if necessary, may be converted to taxol by semisynthesis.

Taxanes from somatic embryos and embryogenic calli derived from zygotic embryos were identified by HPLC, cytotoxicity testing and EL
Completed by ISA with taxol and 6 taxol derivatives supplied by NCI as standard.

The invention will now be described in more detail by way of examples,
This should not be construed as limiting the disclosure of the examples.

Example 1 Taxol was recovered from yew zygote embryos.

Seeds were harvested from excellent trees of Taxus cuspitdata. Embryos were dried in a vacuum oven at about 40 ° C. for about 3 days to a moisture content of less than 10% and weighed. The embryos were then frozen in liquid nitrogen and crushed. The crushed embryo was placed in an incubator at about 28 ° C. containing about 100 times the weight of methanol for about 7 times.
Dipped for days. The resulting extract was filtered through a membrane filter (pore size, 0.2 μm) and analyzed for taxol content using HPLC.

Bark and leaves collected from the same tree were treated according to the same method described above and analyzed for taxol content.

 Table 2 shows the results.

Example 2 Somatic embryos were produced from embryos by using the PEDC pattern.

7 seeds from selected genotypes of Taxus cuspidata
Dipped in 0% ethanol and 2% sodium hypochlorite for 50 seconds and 24 hours, respectively.
Surface sterilized by rinsing with sterile distilled water ~ 5 times.

As the culture medium, was used 2.0ppm of NAA, kinetin and 0.5ppm of 2,4-D supplemented with mB ₅ medium 0.5ppm (having components and composition shown in Table 1 above). The in vitro culture was performed for 8 weeks in a growth room adjusted to 26 ° C.

During the first 8 weeks of culture, the embryos grew larger and calli were induced from the surface of most embryos. Excised green spots in the callus were cultured for about 3 months plant growth regulators in order to induce somatic embryos nothing in mB ₅ medium without putting.

Example 3 Somatic embryos were produced from zygotic embryos by using the IEDC pattern.

Taxus cuspidata seeds were sterilized according to the method of Example 2, and then embryos were isolated therefrom. 2 ppm embryo
The cells were cultured in Durzan medium supplemented with 2,4-D at 23 to 28 ° C for 2 weeks. After two weeks of culture, all embryos were totally covered with potassium derived therefrom. After 4 weeks of cultivation, the potash volume reached tens of times the original volume.

To avoid callus growth inhibition or death due to phenols secreted by the callus itself, callus subcultures were performed using the same medium supplemented with about 1% polyvinylpolypyrrolidone. After 8 weeks of culture, when the average diameter of the potassium has reached 1～1.5Cm, transferred callus mB ₅ medium containing no plant growth regulator, wherein callus was grown to induce somatic embryos. Root differentiation was observed in some embryos.

Example 4 The sterilized zygotic embryos in Example 2 and the somatic embryos in Examples 3 and 4 were used as starting materials for inducing embryogenic callus culture.

The material was 2.0 ppm NAA and 1.0 ppm to induce callus.
about 2 supplemented ppm of kinetin mB in ₅ medium at 23 to 28 ° C..
Cultured for 5 months. After completion of callus induction, subculture was performed for about 6 weeks using the same medium containing 3 ppm of zeatin to obtain a green-yellowish embryoid body.

Culturing the embryoid bodies in the same medium containing 3 ppm of 2,4-D at 26 ° C. for about 2 months under 16/8 (light / dark) time conditions;
It produced 100,000 times the embryogenic callus of the original embryo. The resulting embryogenic callus is composed of various types of yew
Morphologically different from normal calli derived from T. cervix tissues, and sometimes appeared similar to zygotic embryos.

However, the surface of embryogenic calli consisted of various colors ranging from red, yellow, dark green, light brown, dark brown and black. Each colored cell mass was sliced with a blade to create a small cell mass, which was then subcultured in liquid medium for 2-4 weeks. Large cell clumps (cell clu) from liquid culture
mp) after removal of single cells and / or small cell masses
Seeds were placed in plastic Petri dishes containing 20 ml of culture medium. The method adopted was liquid plating (liquid plati
ng) and selection of differently colored cell lines was performed after 4 weeks of culture. These visual selections were made to obtain different colored cell lines.

Example 5 Somatic embryo obtained in Example 2 or 3, or Example 4
The embryogenic potash obtained in the above was dried at 25 ° C. and dissolved in 1 μl of methylene chloride. Add 1 μl of distilled water and mix the mixture
After stirring for 10 seconds, centrifugation (25,000 × g) was performed. The precipitate was dried at 25 ° C., dissolved in 50 μl of methanol, and filtered through a 0.2 μm filter to obtain an extract.

The extract was analyzed by HPLC using a reversed-phase micropore column using a standard curve of an external standard (correlation coefficient 0.999). The HPLC results show that the peaks of the standard taxanes and the taxane contained in the extract have the same retention time (14.3
Minutes).

Example 6 The somatic embryos in Examples 2 and 3 and the embryogenic callus in Example 4 were extracted as follows to produce taxanes.

0.5 g each of the somatic embryos in Examples 2 and 3 and the embryogenic callus in Example 4 were placed in a centrifuge tube,
2 μl of hexane was added. The mixture was mixed well using a glass rod and stored at -20 ° C for 12 hours.
Centrifuged at 25,000 xg for 20 minutes.

A mixed solution of methanol: ethylene chloride was added to the precipitate, and the mixture was subjected to ultrasonic treatment using a sonicator (Branson 250). The resulting solution was centrifuged at 25,000 xg,
The precipitate was dried at 60 ° C. until dry to obtain an extract.

The extracts were analyzed for taxol content and the results were as follows. The somatic embryo extract of Example 2 contained 0.21 to 0.27 mg / g of dry cells (total taxol content was 0.5 to 1.2 mg) and the somatic embryo extract of Example 3 contained 0.20 to 0.27 mg (total taxol content 0.55
The extract of embryogenic callus of Example 4 contained 0.23-0.28 mg (total taxol content was 0.6-1.4 mg).

Example 7 For mass production of embryogenic callus, the embryogenic callus of Example 4 was cultured using impeller-type bioreactase.

Embryogenic callus of Example 4, 10% PCV Erlenmeyer flask 250ml containing the liquid mB ₅ medium 50ml containing 2,4-D for 2ppm
(Packed cell volume). 18 at 25-28 ° C
After culturing under sterile conditions for days, the stationary phase (stationary p.
hase).

The culture was placed in a 5 liter impeller-type bioreactor containing MS medium (production medium) supplemented with 2 ppm NAA. Cultures were maintained at 25-28 ° C for 30 days under aerobic conditions. After 30 days of culture, the culture broth was allowed to settle for 24 hours, allowing the cells to settle, and then separating the cells from the culture medium. A Partur pipette was used to thoroughly remove the culture medium from the cells.

The cells and culture medium thus isolated were extracted as described in Example 5 to produce taxol and its derivatives.

HPLC results showed that the peaks of standard taxol and its derivatives (FIG. 1 (A)) and the peaks of taxol and its derivatives contained in the extract appeared in the same retention period, indicating that these compounds Are the same.

The cells contained 0.09 mg taxol / g dry cells, while the culture medium was calculated to contain about 8 mg taxol / 1.

To examine the effect of the type of the culture medium for Example 8 Taxol production, the cultures of Example _{7 MS, mB 5, WPM,} DKW, Durza
Inoculated in various culture media such as n, White, LP, GD, B5, DCR or SH media. All tested media contain 2.0 ppm
Complementing NAA, microenvironmental conditio
Other factors, including n), were adjusted equivalently. Culture period is 20
Reduced to days.

FIG. 4 shows the results. As can be seen from Figure 4, the production of taxol was significantly influenced by the type of culture medium, it gave the highest taxol production MS and mB ₅ medium.

Example 9 Various elicitors may be added to the production medium to increase taxol and taxane production. The effect of the elicitor on taxol production was evaluated as follows.

(1) In the case of the present invention, Postal which is a bacterium produced in Taxus species is added to a production medium as an elicitor for taxol production.
An extract of otiopsis sp. was added.

Plant tissues, such as seeds and inner bark, were soaked in 70% ethanol, surface sterilized using 15% H ₂ O ₂ for 15 minutes, and then soaked again in 70% ethanol. The tissue was rinsed four or more times with sterile water to remove residual reagent. The tissue thus surface-sterilized is used as a medium: malt extract agar medium (malt extract 20.0 g, peptone 5.0 g,
Agar 15.0 g and distilled water 1 liter), growth agar medium (glucose 40 g, bacto soyton 10)
g, sodium acetate 1g, sodium benzoate 50mg, agar 2
0.0 g and 1 liter of distilled water) and water medium (agar 2
0.0g and 1 liter of distilled water), and 12/12 (dark /
Light) maintained for hours of irradiation.

If Pestalotiopsis sp. Bacteria appear within 3 days of culture,
Further, the bacteria were added to a malt extract and a growth agar medium (yeast extract 3 g, bactosoytone 5 g, MgSO ₄ 0.5 g, glucose 5 g,
Sucrose (10 g / L) for 4 days. At harvest, the culture broth was centrifuged to separate cells from the culture medium. The cells were dried, ground and extracted with methanol to obtain the carbohydrate fraction.

The carbohydrate extract of Pestalotiopsis sp. Thus obtained was added to the production medium to a concentration of 1 ppm, and cultivation and analysis were carried out according to the same method as in Example 7. Table 3 shows the results.

(2) Female gametophyte extract 2 ml / L, phenylalanine 100 mM
Alternatively, the method of Example 7 was repeated except that 1 ppm of gibberellic acid was added as an elicitor to the production medium. Table 3 shows the results.

Example 1 Taxol was demonstrated from a yew tree embryo culture by a cytotoxicity test using rat cancer cells.

Cytotoxicity tests with rat cancer cells are usually based on the fact that taxol can selectively kill cancer cells during metaphase. Republic of Korea, Gyeonggi Province,
Rat carcinoma cells provided by Pacific Research Institute of Singapore were cultured in a plastic incubator containing animal cell culture medium. When cell division occurs early in the culture, one drop of the extract from Example 7 is applied to one incubator (treatment)
, While no extract was added to another incubator (control). Thereafter, cell division and the survival rate of each cancer cell were examined. The results are shown in FIGS. 2 (A) and 2 (B).

As can be seen from FIGS. 2 (A) and 2 (B), the control group showed active growth of cancer cells (FIG. 2).
(A)) On the other hand, the treatment group showed death of cancer cells (FIG. 2).
(B)). Cancer cell death appears 3 hours after treatment and is approximately 24 hours.
After hours, it was completed. Therefore, no measurement of cell division or viability was required.

Experimental Example 2 Identification of taxanes from yew tree embryo culture
This was accomplished by ELISA (enzyme-linked immunosorbent assay) using a monoclonal antibody as follows.

The TA01 kit that specifically reacts with taxol and the TA03 kit that specifically reacts with taxanes were used in this experiment.
Purchased from. Taxol and taxane antigens are PBS
(Phosphate buffered saline) diluted 1/100 and distributed to 100 l ELISA plate of diluent. After incubating for 1 hour at 25 ° C., plate is washed at least 4 times with TBS
-Wash with (wash buffer) and add 50 μl of PBST
(Phosphate buffered saline Tween) was added. Taxol standards, taxane standards or the extract of Example 7 were distributed into the wells by making serial three-fold dilutions.

Taxol antibody and taxane antibody
It was diluted 1/100 and 1 / 100-fold and 50 μl of each dilution was dispensed. After 1 hour incubation at 25 ° C., the plates were washed four times with wash buffer. 1 / 2000-fold diluted in PBST
100 μl of HRP (horseradish peroxidase) was dispensed into the wells and incubated at 25 ° C. for 1 hour, followed by 4 washes with wash buffer. 200 μl of OPD (o-
(Phenylenediamine) was added, and the mixture was further incubated at 25 ° C. for 1 hour to develop color. The absorbance at 490 nm was measured using an ELISA reader. The results are shown in FIG. 3 (A) for the taxol standard substance and in FIG. 3 (B) for the culture medium of Example 7, thereby demonstrating the taxol activity of the yew species embryo culture. It is also to be understood that the above detailed description is for the purpose of illustration only and may be modified and changed without departing from the spirit and scope of the invention.

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Song, Sun-ho Korea 441-090 Kunggi-do Swon-city Kwon-sun-k Codeung-dong 38-1 (56) References International Publication 92/13961 (WO, A1) (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12P 17/00-17/18 C12N 5/00-5/04 WPI (DIALOG) BIOSIS (DIALOG)

Claims (11)

(57) [Claims] A somatic embryo derived from callus derived from the zygote embryo or a culture medium thereof, or an embryogenic callus formed after passing through the somatic embryo or a germ callus thereof. A method for producing taxol or a derivative thereof by recovering taxol or a derivative thereof from a culture medium. 2. Taxol or a derivative thereof is cultured from a zygote of a yew species and cultured from an embryogenic callus or a culture medium thereof after passing through a somatic embryo derived from a callus derived from the zygote. A method for producing taxol or a derivative thereof by collecting, comprising: (a) preparing a zygote embryo from seed of a yew species and disinfecting it; (b) inducing callus from the embryo The disinfected zygote embryos were inoculated and cultured on a solid medium, and then cultured for callus growth in a similar medium further supplemented with a compound capable of preventing callus growth inhibition by phenolic compounds. (C) in order to induce somatic embryos,
Cultured in a solid medium not containing a plant growth regulator; (b) containing 1 to 4 ppm of NAA and 0.5 to 2 ppm of kinetin for inducing embryogenic callus formed after passing through the somatic embryo In order to culture the somatic embryo in a solid medium and form an embryoid body on the surface of the embryogenic callus,
Culturing in a similar medium containing 55 ppm cytokinin and then culturing embryoid bodies in a similar medium containing 2-10 ppm 2,4-D to induce the embryogenic callus; e) culturing the resulting embryogenic callus in a liquid culture; and (f) recovering taxol or a taxol derivative from the culture medium and the cells.
The method described in the section. 3. The method according to claim 2, wherein the step (c) is performed by inducing a somatic embryo by using a PEDC (pre-embryonic development determining cell) or IEDC (induced embryonic development determining cell) method. The described method. 4. The method according to claim 2, wherein said step (e) comprises a growth step using a growth medium and a taxol production step using a production medium. 5. The variant growth medium containing 2,4-D for 2~4ppm Gamborg B ₅ (mB ₅₎ is medium, the production medium 1~2ppm
The method according to paragraph 4 claims a MS or mB ₅ medium supplemented with the NAA. 6. A (corrected) production medium wherein a carbohydrate fraction extract from a bacterium which occurs in Taxus species as an elicitor, an extract of a female gametophyte of Taxus species, phenylalanine or gibberellin 3 to enhance taxol-producing ability. 5. The method according to claim 4, comprising: 7. The method according to claim 6, wherein the fungus is Pestalotiopsis sp. 8. The method according to claim 2, wherein the embryogenic callus in step (d) is extracted to produce taxol or a derivative thereof. 9. The method according to claim 2, wherein the compound supplemented to the medium for callus growth in the step (b) is activated carbon or polyvinylpolypyrrolidone. 10. The method according to claim 2, wherein said cytokinin is benzylamine purine, N-isopentenylaminopurine, kinetin or zeatin. 11. The method according to any one of claims 1 to 10, wherein the yew species is Taxus cuspidata.