HK1150059B - Method for cellular tissue multiplication from jatropha curcas - Google Patents

Description

Method for expanding cell tissue from Jatropha curcas

Technical Field

The present invention relates to the expansion of cells derived from oil-bearing plant seeds, and the obtaining of oil from the resulting cells.

Background

Seeds of oil-bearing plants have been identified as a source for producing oil that can be used for such purposes as biodiesel.

One example is the Jatropha curcas (Jatropha curcas) plant, which has oil-containing seeds. In addition to use in medicine, veterinary medicine, soap making, and the like, the oil is also considered suitable as a high-quality biodiesel. Oil derived from jatropha curcas seeds has similar physicochemical properties and output characteristics as diesel used in engines (see U.S. patent application publication No.2008/0194026a1 to Murali et al, paragraph 0008-.

Thus, in the prior art, micropropagation methods for producing jatropha plants from organs, tissues, cells or protoplasts have been described. (see Murali et al, paragraphs 0014, 0017 and 0024). The resulting plants can be used as a seed source for oil production (see, e.g., Shuyi Qiu et al, Chinese (CN) patent publication No.11225416A, abstract).

Unfortunately, the possibility of producing oil from jatropha curcas seeds in large yields for biodiesel use is limited because of the enormous land area required to harvest plants produced by micropropagation or traditional breeding.

The present invention provides a method for producing oil from jatropha curcas without the need for plant cultivation. Thus, the method of the invention does not require the use of land area for the production of oil from jatropha curcas or from other oil-bearing plants.

Disclosure of Invention

The present invention provides a method for producing oil from cells expanded from jatropha curcas seed tissue, wherein the method is also applicable to other oil-containing plant seeds. The method includes obtaining an explant from cotyledons of a jatropha curcas seed, inoculating the explant into a medium containing an enzyme that disrupts intercellular binding, producing expanded isolated cells by incubation performed under agitation, and extracting oil useful as biodiesel from the expanded cells obtained from isolated cells obtained from cotyledonary tissue of the jatropha curcas seed.

Specifically, the method of the present invention comprises: obtaining an explant from a jatropha curcas seed; placing the explant derived from jatropha curcas seed in a liquid medium; disrupting cell-to-cell binding of tissue explants from jatropha curcas seeds in said culture, wherein said explants give rise to independent cells; incubating the culture medium with isolated cells produced from explants derived from jatropha curcas seeds for a determined time, wherein the isolated cells expand within the determined time; and extracting oil from cells expanded from isolated cells produced from explants derived from Jatropha curcas seeds.

In one aspect of the method of the invention, the explant is obtained from jatropha curcas seed by a method comprising the steps of: disinfecting the jatropha curcas seeds with ethanol, wherein the seeds are bathed with ethanol; hydrating the sterilized jatropha curcas seeds with a paper wetted with sterile water, wherein the jatropha curcas seeds are wrapped with the paper wetted with sterile water; sterilizing the hydrated jatropha curcas seeds with sodium hypochlorite, wherein the hydrated jatropha curcas seeds are bathed with sodium hypochlorite; removing the skin of the sterilized Jatropha curcas seed to release the seed cotyledon from the Jatropha curcas seed; and obtaining an explant from the jatropha curcas seed cotyledons.

The method of the invention can be applied to any oil-bearing plant. The method suitable for any oil-bearing plant comprises:

A. obtaining explants from oil-bearing plant seeds;

B. placing an explant from the seed in a culture medium;

C. disrupting cell-to-cell binding in the culture medium of explant tissue from the seed, wherein the explant produces isolated cells;

D. incubating the culture medium with individual cells produced from an explant derived from the seed for a determined time, wherein the individual cells expand within the determined time; and

E. extracting oil from cells expanded from the isolated cells produced from the explant derived from the seed.

In another aspect of the method of the present invention, preferably, the medium contains at least NH₄NO₃、CaNO₃、CuSO₄、MnSO₄、ZnSO₄、H₃BO₃、KH₂PO₄、Na₂MoO₄、EDTA、FeSO₄、CaCl₂、CaCO₃、NaC₆H₇O₇(sodium citrate), MgSO₄、K₂SO₄Thiamine, glycine, inositol, niacin, pyridoxine, biotin, glutamine, naphthylacetic acid, zeatin and a carbon source. In one aspect of the culture medium of the methods of the invention, the culture medium can comprise a medium selected from CaNO₃And KNO₃A salt.

In one aspect of the medium of the method of the invention, the medium may contain a medium selected from CaCl₂And salts of KCl.

In another aspect of the culture medium of the method of the invention, the culture medium may contain a hormone selected from the group consisting of indoleacetic acid (IAA), naphthaleneacetic acid (NAA) and indolebutyric acid (IBA).

In one aspect of the medium of the method of the present invention, the medium may contain a hormone selected from the group consisting of kinetin, Benzyladenine (BA), Gibberellin (GA), and zeatin.

In one aspect of the medium of the method of the invention, the medium may contain a carbon source selected from sucrose, fructose and glucose.

In another aspect of the culture medium of the method of the invention, cellulase, pectinase and hemicellulase are added to the culture medium, wherein the cellulase, pectinase and hemicellulase disrupt the intercellular bonds of the tissue from the explants of the jatropha curcas seed in the culture medium.

In another aspect of the method of the present invention, the oil is extracted from a medium containing independent cells expanded from independent cells produced from an explant of a crazy tree seed by the steps comprising: adding an organic solvent; carrying out ultrasound; centrifuging; taking out the upper layer; and evaporating and drying the solvent.

The invention also provides a culture medium, wherein the culture medium contains NH₄NO₃、CaNO₃、CuSO₄、MnSO₄、ZnSO₄、H₃BO₃、KH₂PO₄、Na₂MoO₄、EDTA、FeSO₄、CaCl₂、CaCO₃、NaC₆H7O₇(sodium citrate), MgSO₄、K₂SO₄Thiamine, glycine, inositol, niacin, pyridoxine, biotin, glutamine, naphthylacetic acid, zeatin, and a carbon source.

In one aspect of the culture medium of the present invention, the culture medium contains cellulase, pectinase and hemicellulase, wherein the cellulase, pectinase and hemicellulase disrupt the intercellular bonds of the tissue from the explant of the jatropha curcas seed in the culture medium.

In one aspect of the media of the present invention, the media may comprise a medium selected from the group consisting of CaNO₃And KNO₃A salt.

In one aspect of the medium of the present invention, the medium may contain CaCl₂And salts of KCl.

In one aspect of the medium of the present invention, the medium may contain a hormone selected from the group consisting of indoleacetic acid (IAA), naphthylacetic acid (NAA) and indolebutyric acid (IBA).

In one aspect of the medium of the present invention, the medium may contain a hormone selected from the group consisting of kinetin, Benzyladenine (BA), Gibberellin (GA), and zeatin.

In one aspect of the medium of the present invention, the medium may contain a carbon source selected from sucrose, fructose and glucose.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the embodiments of the present invention and the appended claims.

Detailed Description

The method of the invention comprises the following steps: obtaining an explant from a jatropha curcas seed; placing explants derived from Jatropha curcas seeds in a culture medium; disrupting cell-to-cell binding of tissue explants from jatropha curcas seeds in the culture medium, wherein the explants give rise to independent cells; incubating the culture medium with isolated cells produced from explants derived from jatropha curcas seeds for a determined time, wherein the isolated cells expand within the determined time; and extracting oil from cells expanded from isolated cells produced from explants derived from Jatropha curcas seeds.

In a preferred form of the method of the invention, the preferred incubation time is at least 3 days from the time the explant is placed in culture. In a more preferred form, the incubation time is at least 8 days from the time the explant is placed in culture. In a more preferred form, the incubation time is at least 14 days from the time the explant is placed in culture. The incubation is preferably carried out in the dark at room temperature under constant stirring.

The incubation can also be performed under illumination with a light source emitting light of a defined wavelength, such as infrared light, or a light source emitting light of a wavelength optimal for cell expansion from a particular oleaginous plant seed.

Once incubated in the medium for at least 3 daysAfter which the cell-cell binding is disrupted and the isolated cells produced from the explants derived from the jatropha curcas seed can be used as a source of cells for re-inoculation into fresh medium, with repeated re-inoculation into fresh medium, thereby resulting in continuous expansion of the isolated cells. The preferred amount of isolated cells to be re-inoculated into fresh medium is 1 to 2X 10⁵Cells/ml, the fresh reseeding medium was incubated for a defined time until the cells reached an expansion stationary phase.

In one aspect of the method of the invention, the explant is obtained from jatropha curcas seed by a method comprising the steps of: disinfecting the jatropha curcas seeds with ethanol, wherein the seeds are bathed with ethanol; hydrating the sterilized jatropha curcas seeds with a paper wetted with sterile water, wherein the paper wetted with sterile water encapsulates the jatropha curcas seeds; sterilizing the hydrated jatropha curcas seeds with sodium hypochlorite, wherein the hydrated jatropha curcas seeds are bathed with sodium hypochlorite; removing the outer skin of the sterilized Jatropha curcas seed to release the seed cotyledon from the Jatropha curcas seed; and obtaining an explant from the jatropha curcas seed cotyledons.

For the purposes of the present application, the term explant refers to a portion of the cotyledon from the jatropha curcas seed. In a preferred form, the explants obtained from the cotyledons of the jatropha curcas seeds consist of a transection of the cotyledons in layers 1 to 2 mm thick. The explant may also be other types of cotyledon parts of different shapes from jatropha curcas seeds or other oil-containing plant seeds. In a preferred form of the invention, the amount of the explant may be 1 to 7 g per 100 ml of medium.

The method of the invention can be applied to any oil-bearing plant. The method suitable for any oil-bearing plant comprises:

A. obtaining explants from oil-bearing plant seeds;

B. placing an explant from the seed in a culture medium;

C. disrupting cell-to-cell binding in the culture medium of explant tissue from the seed, wherein the explant produces isolated cells;

D. incubating the culture medium with individual cells produced from an explant derived from the seed for a determined time, wherein the individual cells expand within the determined time; and

E. extracting oil from cells expanded from the isolated cells produced from the explant derived from the seed.

The term "oleaginous plant" refers to any plant from which oil can be extracted from its seeds or fruits, which in some cases is edible, in other cases is useful in industry, and in yet other cases is useful in medicine. The oil-containing plants include:

soybean (Glycine max) (Soybean)

African oil palm (Elaeis guineensis) (African palm)

American oil palm (Elaeis oleifera) (American oil palm)

Peanut (Arachis hypogaea) (peanut)

Sunflower (Helianthus annuus) (sunflower)

Corn (Zea mays) (corn)

Flax (Linum usittissimum) (flax seed)

Rape (Brassica napus) (Brassica, rape, rapeseed or nabicol)

Olea europaea (Olea europaea L.) (Olive)

Castor (Ricinus communis) (castor oil)

Sesame () (sesame)

Jojoba (Simmondsia chinensis) (jojoba oil)

Tung-oil tree (Vernicia fordii) (tung tree)

Mountain apricot (Prunus dulcis) (almond)

Safflower (Carthamus tinctorius) (safflower or saffron (alazor))

Upland cotton (Gossypium hirsutum) (cotton)

Moringa oleifera (Moringa oleifera) (drumstick tree)

Wheat (Triticum aestivum) (wheat)

And plants containing oil in all kinds of seeds.

In other aspects of the methods of the invention, preferably, the medium contains at least NH₄NO₃、CaNO₃、CuSO₄、MnSO₄、ZnSO₄、H₃BO₃、KH₂PO₄、Na₂MoO₄、EDTA、FeSO₄、CaCl₂、CaCO₃、NaC₆H₇O₇(sodium citrate), MgSO₄、K₂SO₄Thiamine, glycine, inositol, niacin, pyridoxine, biotin, glutamine, naphthylacetic acid, zeatin, and a carbon source.

In one aspect of the culture medium of the methods of the invention, the culture medium can comprise a medium selected from CaNO₃And KNO₃A salt.

In one aspect of the medium of the method of the invention, the medium may contain a medium selected from CaCl₂And salts of KCl.

In one aspect of the medium of the method of the invention, the medium may contain a hormone selected from the group consisting of indoleacetic acid (IAA), naphthaleneacetic acid (NAA) and indolebutyric acid (IBA).

In one aspect of the medium of the method of the present invention, the medium may contain a hormone selected from the group consisting of kinetin, Benzyladenine (BA), Gibberellin (GA), and zeatin.

In one aspect of the medium of the method of the invention, the medium may contain a carbon source selected from sucrose, fructose and glucose.

In one aspect of the culture medium of the method of the invention, cellulase, pectinase and hemicellulase are added to the culture medium, wherein the cellulase, pectinase and hemicellulase disrupt the intercellular bonds of the tissue from the explants of the jatropha curcas seed in the culture medium. The intercellular binding of the explant tissue from the jatropha curcas seed can be disrupted by other enzymatic or non-enzymatic processes.

In a preferred form, the media of the invention have the following ranges of concentrations of the components:

NH₄NO₃ 100-500mg/L

CaNO₃or KNO₃ 200-400mg/L

CuSO₄ 0.1-5mg/L

MnSO₄ 10-40mg/L

ZnSO₄ 20-50mg/L

H₃BO₃ 10-20mg/L

KH₂PO₄ 50-200mg/L

Na₂MoO₄ 0.1-5mg/L

EDTA (ethylene diamine tetraacetic acid) 10-50mg/L

FeSO₄ 10-50mg/L

CaCl₂Or KCl 50-100mg/L

CaCO₃ 30-50mg/L

NaC₆H₇O₇(sodium citrate) 0.1-10mg/L

MgSO₄ 50-300mg/L

K₂SO₄ 700-1500mg/L

Thiamine 0.5-3mg/L

0.5-3mg/L of glycine

50-200mg/L of inositol

Nicotinic acid 0.1-10mg/L

Pyridoxine 0.1-10mg/L

Biotin 0.1-10mg/L

20-50mg/L of glutamine

IAA or NAA or IBA 0.5-10mg/L

Zeatin or kinetin or BA or GA 0.5-10mg/L

Sucrose or glucose or fructose 30000-100000mg/L, and

hemicellulase and pectinase and cellulase 3000-10000mg/L

In a preferred form of the method of the invention, the EDTA is the disodium salt. Monosodium EDTA may also be used in the methods of the invention.

In a preferred form of the method of the invention, the inositol is myo-inositol. In a preferred form of the method of the invention, the pH of the medium is adjusted to a value of 4.8 to 6.5.

In another aspect of the method of the present invention, the oil is extracted from the culture medium containing the isolated cells expanded from the isolated cells produced from the explant from the jatropha curcas seed by a process comprising the steps of: adding an organic solvent; carrying out ultrasound; centrifuging; taking out the upper layer; and evaporating and drying the solvent.

For jatropha or any other oil-containing plant, oil extraction is performed from cells expanded from individual cells produced from explants from seed cotyledons when the cell mass reaches stationary phase in the culture medium. An organic solvent is added to the culture medium containing the expanded cells. The preferred organic solvent is hexane, but other solvents such as isopropanol or ethanol, etc. may also be used.

The walls of the individual cells are disrupted when the solvent is mixed, and the medium and the individual cells are sonicated to release the oil from the cells. The mixture of solvent and medium containing the disrupted cells and released oil is centrifuged to separate the mixture into two phases: an upper phase and a lower phase. The upper phase contains the solvent and released oil, and the lower phase contains the cell residue and culture medium. The upper phase, containing the solvent and the oil, is separated and the solvent of said phase is evaporated by rotary evaporation and heating, thus achieving the purification of the oil.

The invention also provides a culture medium, wherein the culture medium contains NH₄NO₃、CaNO₃、CuSO₄、MnSO₄、ZnSO₄、H₃BO₃、KH₂PO₄、Na₂MoO₄、EDTA、FeSO₄、CaCl₂、CaCO₃、NaC₆H₇O₇(sodium citrate), MgSO₄、K₂SO₄Thiamine, glycine, inositol, niacin, pyridoxine, biotin, glutamine, naphthylacetic acid, zeatin, and a carbon source.

In one aspect of the culture medium of the present invention, the culture medium contains cellulase, pectinase and hemicellulase, wherein the cellulase, pectinase and hemicellulase disrupt the intercellular bonds of the explant tissue from the jatropha curcas seed in the culture medium.

In one aspect of the culture medium of the methods of the invention, the culture medium can comprise a medium selected from CaNO₃And KNO₃A salt.

In one aspect of the medium of the method of the invention, the medium may contain a medium selected from CaCl₂And salts of KCl.

In one aspect of the medium of the present invention, the medium may contain a hormone selected from the group consisting of indoleacetic acid (IAA), naphthylacetic acid (NAA) and indolebutyric acid (IBA).

In one aspect of the medium of the present invention, the medium may contain a hormone selected from the group consisting of kinetin, Benzyladenine (BA), Gibberellin (GA), and zeatin.

In another aspect of the medium of the present invention, the medium may contain a carbon source selected from sucrose, fructose and glucose.

In a preferred form, the media of the invention have the following ranges of concentrations of the components:

NH₄NO₃ 100-500mg/L

CaNO₃or KNO₃ 200-400mg/L

CuSO₄ 0.1-5mg/L

MnSO₄ 10-40mg/L

ZnSO₄ 20-50mg/L

H₃BO₃ 10-20mg/L

KH₂PO₄ 50-200mg/L

Na₂MoO₄ 0.1-5mg/L

EDTA (ethylene diamine tetraacetic acid) 10-50mg/L

FeSO₄ 10-50mg/L

CaCl₂Or KCl 50-100mg/L

CaCO₃ 30-50mg/L

NaC₆H₇O₇(sodium citrate) 0.1-10mg/L

MgSO₄ 50-300mg/L

K₂SO₄ 700-1500mg/L

Thiamine 0.5-3mg/L

0.5-3mg/L of glycine

50-200mg/L of inositol

Nicotinic acid 0.1-10mg/L

Pyridoxine 0.1-10mg/L

Biotin 0.1-10mg/L

20-50mg/L of glutamine

IAA or NAA or IBA 0.5-10mg/L

Zeatin or kinetin or BA or GA 0.5-10mg/L

Sucrose or glucose or fructose 30000-100000mg/L, and

hemicellulase, pectinase and cellulase 3000-5000mg/L

In a preferred form of the medium of the invention, EDTA is the disodium salt. Monosodium EDTA may also be used in the methods of the invention.

In a preferred form of the medium of the invention, the inositol is inositol.

In a preferred form of the medium of the invention, the pH is adjusted to a value of 4.8 to 6.5.

While this specification sets forth preferred embodiments of the invention, additional changes in the form and arrangement of the parts herein may be made without departing from the concepts and the underlying principles as covered by the claims.

Examples

Cleaning and washing mature jatropha curcas seeds with water and soap in advance, and disinfecting the seeds by using an ethanol dipping bath; then, hydrating the seeds by wrapping them with paper wetted with sterile water; subsequently, the seeds were sterilized with a 3% sodium hypochlorite bath; then, removing the skin of the seeds by a knife; explants or cross-sectioned lamellar parts 1 to 2 mm thick were cut from the cotyledons of the resulting jatropha curcas seeds.

1 to 3 grams of explants of jatropha curcas seed cotyledons were placed in 100 ml of medium with the following composition and concentration:

NH₄NO₃ 400mg/L

CaNO₃ 383mg/L

CuSO₄ 0.25mg/L

MnSO₄ 22.3mg/L

ZnSO₄ 8.6mg/L

H₃BO₃ 6.2mg/L

KH₂PO₄ 170mg/L

Na₂MoO₄ 0.25mg/L

Na₂EDTA (ethylene diamine tetraacetic acid) 37.3mg/L

FeSO₄ 27.85mg/L

CaCl₂ 72.5mg/L

CaCO₃ 20mg/L

NaC₆H₇O₇(sodium citrate) 0.5mg/L

MgSO₄ 180.7mg/L

K₂SO₄ 990mg/L

Thiamine 1mg/L

Glycine 2mg/L

Inositol 100mg/L

Nicotinic acid 0.5mg/L

Pyridoxine 0.5mg/L

Biotin 1mg/L

Glutamine 30mg/L

IAA 2mg/L

Kinetin 1mg/L

Sucrose 50000mg/L, and

pectinase and cellulase 5000mg/L

The pH of the medium is adjusted to 5.8 with 0.1N HCl and/or NaOH solution.

The medium was incubated at room temperature in the dark under constant stirring for 15 days, at which time the conversion of the explant tissue into individual expanded cells was observed at a density of about 2X 10⁵Individual cells/ml.

The cells were separated from the medium, inoculated into 100 ml of fresh medium of the same composition and concentration, and further incubated in the dark under constant stirring for about 10 days, at which time the cells expanded to reach stationary phase (about 2X 10)⁶Individual cells/ml).

100 ml of hexane was added to a solution containing hexane having a density of about 2X 10⁶Individual cells/ml of individual cells in 100 ml of medium. The resulting mixture was sonicated for 90 minutes. The mixture was then centrifuged at 2500rpm for 30 minutes. After centrifugation, an upper phase and a lower phase appeared. The upper phase was separated, transferred to a receiver, rotary evaporated at 180rpm at 70 ℃ and then placed on a 75 ℃ oven for 1 hour. The weight of the oil obtained was 1.978 g (yield 19.78 g/L).

The oil was analyzed and the results are shown in the following table:

table 1: fatty acid determination of the composition of the oil samples.

Composition of the oil samples determined (gas chromatography mass spectrometry):

fatty acid retention time (min) composition% (p/p)

Myristic acid 11.7660.26

Azelaic acid 13.1550.15

Palmitic acid 13.46019.96

Palmitoleic acid 13.6980.61

Stearic acid (Estearic) 14.9948.64

Oleic acid 15.18838.46

Linoleic acid 15.55531.35

Eicosanoic acid 16.3870.56

The composition of the oil obtained by the method of the invention from isolated cells derived from the cotyledons of Jatropha curcas seeds is similar to that reported in the literature, for example, as shown in the following Table, which is obtained from Akintoayo et al, Bioresource technology 92(2004) 307-310.

Akintayo's Table 3 (page 309)

Fatty acid composition of seed oil of Parkia biglobbossa (PKBS) (% Parkia biglobbossa)) and Jatropha curcas (JTC (Jatropha curcas) (%)

Fatty acid PKBS JTC

Palmitic acid 27.5 + -0.519.5 + -0.8

Stearic acid (Estearic) 10.5 + -0.46.8 + -0.6

Oleic acid 14.5 +/-0.541.3 +/-1.5

Linoleic acid 44.5 + -1.531.4 + -1.2

Linolenic acid 3.0 +/-0.2-

Saturated acid 3826.3

Unsaturated acid 6272.7

The values shown are the mean of two determinations. + -. standard deviation.

The composition of the oil derived from jatropha curcas obtained by the process of the invention is comparable to the composition of the oil derived from jatropha curcas described in the literature. Thus, the oil obtained from jatropha curcas by the process of the present invention has the characteristics required for high quality biodiesel as described in the literature on jatropha curcas source oils.

Claims (23)

1. A method of producing oil derived from an isolated cell expanded from a plant seed explant, wherein said method comprises:

a. obtaining an explant from the seed;

b. placing an explant from the seed in a culture medium;

c. disrupting cell-to-cell binding in the explant tissue from the seed by adding to the culture medium an enzyme capable of disrupting said cell-to-cell binding, wherein the explant produces individual cells;

d. incubating and expanding in said culture medium individual cells produced from explants derived from said seed; and

e. extracting oil from cells expanded from isolated cells produced from an explant derived from said plant seed.

2. The method of claim 1, wherein the plant seed is an oleaginous plant seed.

3. A method of producing oil derived from isolated cells expanded from jatropha curcas seed explants, wherein the method comprises:

a. obtaining an explant from the jatropha curcas seed;

b. placing the explant from the jatropha curcas seed in a culture medium;

c. disrupting cell-to-cell binding of explant tissue from the jatropha curcas seed by adding to the culture medium an enzyme capable of disrupting the cell-to-cell binding, wherein the explant produces independent cells;

d. incubating and expanding in the culture medium the isolated cells produced by the explants derived from the jatropha curcas seed; and

e. extracting oil from cells expanded from the isolated cells produced from the explant derived from the Jatropha curcas seed.

4. The method of any one of claims 1 to 3, wherein the explant is obtained from a plant seed by a method comprising the steps of:

a. disinfecting plant seeds with ethanol, wherein the seeds are bathed with ethanol;

b. hydrating the sterilized plant seeds with a paper wetted with sterile water, wherein plant seeds are wrapped with the paper wetted with sterile water;

c. sterilizing the hydrated plant seeds with sodium hypochlorite, wherein the hydrated plant seeds are bathed with sodium hypochlorite;

d. removing the skin from the sterilized plant seed to release the seed cotyledon from the plant seed; and

e. obtaining an explant from said plant seed cotyledon.

5. The method of any one of claims 1 to 3, wherein the medium used in steps (b), (c) and (d) comprises at least NH₄NO₃、Ca(NO₃)₂、CuSO₄、MnSO₄、ZnSO₄、H₃BO₃、KH₂PO₄、Na₂MoO₄、EDTA、FeSO₄、CaCl₂、CaCO₃、NaC₆H₇O₇、MgSO₄、K₂SO₄Thiamine, glycine, inositol, niacin, pyridoxine, biotin, glutamine, naphthylacetic acid, zeatin, and a carbon source.

6. The method of any one of claims 1 to 3, wherein cellulase, pectinase and hemicellulase are added to the culture medium, wherein the cellulase, pectinase and hemicellulase disrupt the intercellular binding of explant tissue from the plant seed in the culture medium.

7. The method of claim 5, wherein said medium used in steps (b), (c) and (d) contains Ca (NO)₃)₂And KNO₃A salt.

8. The method of claim 5, wherein said medium used in steps (b), (c) and (d) comprises a medium selected from the group consisting of CaCl₂And salts of KCl.

9. The method of claim 5, wherein said medium used in steps (b), (c) and (d) contains a hormone selected from the group consisting of indoleacetic acid, naphthylacetic acid and indolebutyric acid.

10. The method of any one of claims 7 to 8, wherein the medium used in steps (b), (c) and (d) contains a hormone selected from the group consisting of indoleacetic acid, naphthylacetic acid and indolebutyric acid.

11. The method of claim 5, wherein the medium used in steps (b), (c) and (d) contains a hormone selected from the group consisting of kinetin, benzyladenine, gibberellin and zeatin.

12. The method of any one of claims 7 to 8, wherein the medium used in steps (b), (c) and (d) contains a hormone selected from the group consisting of kinetin, benzyladenine, gibberellin and zeatin.

13. The method of claim 9, wherein the medium used in steps (b), (c) and (d) contains a hormone selected from the group consisting of kinetin, benzyladenine, gibberellin and zeatin.

14. The method of claim 5, wherein the medium used in steps (b), (c) and (d) contains a carbon source selected from the group consisting of sucrose, fructose and glucose.

15. The method of any one of claims 7 to 8, wherein the medium used in steps (b), (c) and (d) contains a carbon source selected from sucrose, fructose and glucose.

16. The method of claim 9, wherein the medium used in steps (b), (c) and (d) contains a carbon source selected from the group consisting of sucrose, fructose and glucose.

17. The method of claim 10, wherein the medium used in steps (b), (c) and (d) contains a carbon source selected from the group consisting of sucrose, fructose and glucose.

18. The method of claim 11, wherein the medium used in steps (b), (c) and (d) contains a carbon source selected from the group consisting of sucrose, fructose and glucose.

19. The method of any one of claims 1 to 3, wherein the incubation time from the time the explant is placed in culture medium is at least 3 days.

20. The method of any one of claims 1 to 3, wherein the incubation time from the time the explant is placed in culture medium is at least 8 days.

21. The method of any one of claims 1 to 3, wherein the incubation time from the time the explant is placed in culture medium is at least 14 days.

22. The method of any one of claims 1 to 3, wherein the incubation time from when the individual cells produced from the explant are re-inoculated into fresh medium is at least 10 days.

23. The method of any one of claims 1 to 3, wherein the oil is extracted from the medium containing said isolated cells expanded from isolated cells produced from explants from plant seeds by a process comprising:

a. adding an organic solvent;

b. carrying out ultrasound;

c. centrifuging;

d. taking out the upper phase; and

e. the solvent was evaporated and dried.