Method for cellular tissue multiplication from jatropha curcas.
The present invention is related to the multiplication of cells derived from seeds of oleaginous plants and the obtention of oils derived from the generated cells. The seeds of oleaginous plants had been identified as sources in the production of oils that could serve, among other uses, as biodiesel. An example is the Consequently, in the prior art, it has been described micro-propagation methods with the purpose of generating Unfortunately, the possibility of oil production derived from the The present invention provides a method for oil generation derived for The present invention provides a method for the production of oil derived from multiplied cells from Specifically, the method of the present invention comprises: Obtaining an explant from the In one aspect of the method of the present invention, the explant is obtained from the The method of the present invention could be applied to any oleaginous plant. Said method adapted to any oleaginous plant comprises: In another aspect of the method of the present invention, preferably, the culture medium contains at least NH4NO3, CaNO3, CuSO4, MnSO4, ZnSO4, H3BO3, KH2PO4. Na2MoO4, EDTA, FeSO4, CaCl2, CaCO3, NaC6H7O7 (sodium citrate), MgSO4, K2SO4, thiamine, glycine, inositol, nicotinic acid, pyridoxine, biotin, glutamine, naftalenacetic acid, zeatin, and a carbon source. In one aspect of the culture medium of the method of the present invention, said culture medium could contain a salt selected from the group that consist of CaNO3, and KNO3. In one aspect of the culture medium of the method of the present invention, said culture medium could contain a salt selected from the group that consist of CaCl2, and KCl. In another aspect of the culture medium of the method of the present invention, said culture medium could contain an hormone selected from the group that consist of indolacetic acid (IAA), naftalenacetic acid (NAA), and indolebutyric acid (IBA). In one aspect of the culture medium of the method of the present invention, said culture medium could contain an hormone selected from the group that consist of kinetine, benziladenine (BA), Gibberelline (GA), and Zeatin. In one aspect of the culture medium of the method of the present invention, said culture medium could contain a carbon source selected from the group that consist of saccharose (sucrose), fructose, and glucose. In another aspect of the culture medium of the method of the present invention, cellulase, pectinase and hemicellulase are added to the culture medium, wherein the cellulase, pectinase and hemicellulase break the tissue intercellular unions of the explants derived from the In one additional aspect of the method of the present invention, the oil is extracted from the culture medium with the individual cells that were multiplied from the individual cells generated from the explants derived from the The present invention also provides a culture medium wherein the culture medium comprises NH4NO3, CaNO3, CuSO4, MnSO4, ZnSO4, H3BO3, KH2PO4, Na2MoO4, EDTA, FeSO4, CaCl2, CaCO3, NaC6H7O7 (sodium citrate), MgSO4, K2SO4, thiamine, glycine, inositol, nicotinic acid, pyridoxine, biotin, glutamine, naftalenacetic acid, zeatin, and a carbon source. In one aspect of the culture medium of the present invention, said culture medium contains cellulase, pectinase and hemicellulase, wherein the cellulase, pectinase and hemicellulase break the intercellular unions of the explants tissue derived from the In one aspect of the culture medium of the present invention, said culture medium could contain a salt selected from the group that consists of CaNO3, and KNO3. In one aspect of the culture medium of the present invention, said culture medium could contain a salt selected from the group that consists of CaCl2, and KCl. In one aspect of the culture medium of the present invention, said culture medium could contain a hormone selected from the group that consists of indolacetic acid (IAA), naftalenacetic acid (NAA), and indolebutyric acid (IBA). In one aspect of the culture medium of the present invention, said culture medium could contain a hormone selected from the group that consists of kinetine, benziladenine (BA), Gibberelline (GA), and Zeatin. In one aspect of the culture medium of the present invention said culture medium could contain a carbon source selected from the group that consists of saccharose (sucrose), fructose, and glucose. Additional objectives and advantages of the present invention will be more evident in the detailed description of the invention and the claims. The method of the present invention comprises: Obtaining an explant from the In the preferred form of the method of the present invention, the preferred incubation time is at least 3 days from the moment in which the explant is put in the culture medium. In a more preferred form, the time of incubation is at least 8 days from the moment in which the explant is put in the culture medium. In a more preferred form yet the time of the incubation is at least 14 days from the moment in which the explant is put in the culture medium. The incubation is preferably performed under constant agitation at room temperature in darkness. The incubation could also be done under illumination under a light source that emits a determined wave length, for example, infrared light, or a light source that emits the wave length that is optimal for multiplication of cells derived from a specific oleaginous plant seed. The individual cells generated from the explants derived from the . In one aspect of the method of the present invention, the explant is obtained from the For purposes of the application of the present invention, the term explant refers to a portion of cotyledon from the The method of the present invention could be applied to any oleaginous plant. Said method adapted to any oleaginous plant comprises: The term "oleaginous plant" is referred to any vegetable from which seed or fruit oil can be extracted, in some cases, edible, in other cases of industrial use and in other cases medicinal. Oleaginous plants include: In other aspect of the method of the present invention, preferably, the culture medium contains at least NH4NO3, CaNO3, CuSO4, MnSO4, ZnSO4, H3BO3, KH2PO4, Na2MoO4, EDTA, FeSO4, CaCl2, CaCO3, NaC6H7O7 (sodium citrate), MgSO4, K2SO4, thiamine, glycine, inositol, nicotinic acid, pyridoxine, biotin, glutamine, naftalenacetic acid, zeatin, and a carbon source. In one aspect of the culture medium of the method of the present invention, said culture medium could contain a salt selected from the group that consist of CaNO3, and KNO3. In one aspect of the culture medium of the method of the present invention, said culture medium could contain a salt selected from the group that consist of CaCl2, and KCl. In one aspect of the culture medium of the method of the present invention, said culture medium could contain an hormone selected from the group that consist of indolacetic acid (IAA), naftalenacetic acid (NAA), and indolebutyric acid (IBA). In one aspect of the culture medium of the method of the present invention, said culture medium could contain an hormone selected from the group that consist of kinetine, benziladenine (BA), Gibberelline (GA), and Zeatin. In one aspect of the culture medium of the method of the present invention, said culture medium could contain a carbon source selected from the group that consist of saccharose (sucrose), fructose, and glucose. In one aspect of the culture medium of the method of the present invention, cellulase, pectinase and hemicellulase are added to the culture medium, wherein the cellulase, pectinase and hemicellulase break the tissue intercellular unions of the explants derived from the In the preferred form, the concentration ranges of the components of the culture medium of the present invention are the following: In the preferred form of the method of the present invention the EDTA is disodic. The monosodic-EDTA can also be used in the method of the present invention. In the preferred form of the method of the present invention the inositol is mio-inositol. In the preferred form of the method of the present invention the culture medium is adjusted for a pH between 4.8 and 6.5. In one additional aspect of the method of the present invention, the oil is extracted from the culture medium with the individual cells that were multiplied from the individual cells generated from the explants derived from the In the case of The walls of the individual cells are broken when the mix of solvent, the culture medium and the individual cells are subjected to sonication, releasing the oil from the cells. The mix of solvent and culture medium that contains the broken cells and the released oil are centrifugated, which separates said mix in two phases, a superior one and an inferior one. The superior phase contains the solvent and the released oil, and the inferior phase contains said cells residues and the culture medium. The superior phase with the solvent and the oil is separated and then, the solvent of said phase is evaporated by roto-evaporation and heating, which results in the purification of oil. The present invention also provides a culture medium wherein the culture medium comprises NH4NO3, CaNO3, CuSO4, MnSO4, ZnSO4, H3BO3, KH2PO4, Na2MoO4, EDTA, FeSO4, CaCl2, CaCO3, NaC6H7O7 (sodium citrate), MgSO4, K2SO4, thiamine, glycine, inositol, nicotinic acid, pyridoxine, biotin, glutamine, naftalenacetic acid, zeatin, and a carbon source. In one aspect of the culture medium of the present invention, said culture medium contains cellulase, pectinase and hemicellulase, wherein the cellulase, pectinase and hemicellulase break the intercellular unions of the explants tissue derived from the In one aspect of the culture medium of the method of the present invention, said culture medium could contain a salt selected from the group that consists of CaNO3, and KNO3. In one aspect of the culture medium of the method of the present invention, said culture medium could contain a salt selected from the group that consists of CaCl2, and KCl. In one aspect of the culture medium of the present invention, said culture medium could contain a hormone selected from the group that consists of indolacetic acid (IAA), naftalenacetic acid (NAA), and indolebutyric acid (IBA). In one aspect of the culture medium of the present invention, said culture medium could contain a hormone selected from the group that consists of kinetine, benziladenine (BA), Gibberelline (GA), and Zeatin. In another aspect of the culture medium of the present invention, said culture medium could contain a carbon source selected from the group that consists of saccharose (sucrose), fructose, and glucose. In the preferred form, the concentration ranges of the components of the culture medium of the present invention are the following: In the preferred form of the culture medium of the present invention the EDTA is disodic. The monosodic-EDTA can also be used in the method of the present invention. In the preferred form of the culture medium of the present invention the inositol is mio-inositol. In the preferred form of the culture medium of the present invention is adjusted for a pH between 4.8 and 6.5. Although the description presents preferred embodiments of the present invention, additional changes may be made in the form and disposition of the parts without deviating from the ideas and basic principles encompassed by the claims. Ripe Between 1 to 3 grams of explants of The culture medium was adjusted to a pH of 5.8 with HCL and/or NaOH 0.1 N solutions. Said culture medium was incubated, under constant agitation, at room temperature, in the darkness for 15 days, when it was observed that explants tissue was converted to individually multiplied cells with a density of 2 x 105 individual cells per milliliter approximately. The cells were separated from the culture medium and were re-inoculated in 100 ml of fresh culture medium of the same composition and concentrations, continuing with the incubation under constant agitation, in the darkness, for approximately 10 more days, when the cells multiplying reached a stationary state (approximately 2 x 106 individual cells per milliliter). 100 ml of hexane were added to the 100 ml of culture medium with individual cells in a density of approximately 2 x 106 individual cells per milliliter. The resulting mix was subjected to sonication for 90 minutes. Then the mix was centrifugated at 2500 rpm for 30 minutes. A superior phase and an inferior phase resulted after centrifugation. The superior phase was separated and was transferred to a recipient which was roto-evaporated at 70°C, and at 180 rpm, and then it was placed in a stove for 1 hour at 75°C. The resulting oil weighted 1.978 grams (resulting yield of 19.78 grams/L). The oil was analyzed with the results as shown in the following table: The composition of the obtained oil from the individual cells derived from cotyledon of the The composition of oil derived from The method of the present invention comprises: Obtaining an explant from the seeds of Jatropha curcas; Putting the explant derived from the seed of Jatropha curcas in a culture medium; Breaking the intercellular unions of the explants tissue, which generates individuals cells; Incubating for a determined time the culture medium with the generated individual cells, that were multiplied; and, Extracting oil from the cells that multiplied from the individual cells generated from the explants derived from the Jatropha curcas seed. A method for production of oil derived from a seed wherein the method comprises:
A. Obtaining an explant from the seed; B. Putting the explant derived from the seed in a culture medium; C. Breaking the intercellular unions of the explants tissue derived from the seed in said culture medium, wherein the explants generate individual cells; D. Incubating for a determined time the culture medium with the individual cells generated from the explants derived from the seed, wherein said individual cells are multiplied within the determined time; and, E. Extracting oil from the cells that multiplied from the individual cells generated from the explants derived from the seed. The method of Claim 1, wherein the seed is an oleaginous plant seed. A method for production of oil derived from a A. Obtaining an explant from the B. Putting the explant derived from the C. Breaking the intercellular unions of the explants tissue derived from the D. Incubating for a determined time the culture medium with the individual cells generated from the explants derived from the E. Extracting oil from the cells that multiplied from the individual cells generated from the explants derived from the The method of Claim 3, wherein the explant is obtained from the A. Disinfecting the B. Hydrating said disinfected C. Sterilizing the hydrated D. Retiring the skin of the sterilized E. Obtaining explants from the The method of Claim 3, wherein the culture medium contains at least NH4NO3, CaNO3, CuSO4, MnSO4, ZnSO4, H3BO3, KH2PO4, Na2MoO4, EDTA, FeSO4, CaCl2, CaCO3, NaC6H7O7 (sodium citrate), MgSO4, K2SO4, thiamine, glycine, inositol, nicotinic acid, pyridoxine, biotin, glutamine, naftalenacetic acid, zeatin, and a carbon source. The method of Claim 3, wherein cellulase, pectinase and hemicellulase are added to the culture medium, wherein the cellulase, pectinase and hemicellulase break the intercellular unions of the explants tissue derived from the J The method of Claim 5, where the culture medium contains a salt selected from the group that consists of CaNO3 and KNO3; and/or a salt selected from the group that consists of CaCl2 and KCl. The method of Claim 5, wherein the culture medium contains a hormone selected from the group that consists of indolacetic acid (IAA), naftalenacetic acid (NAA), and indolebutyric acid (IBA); and/or a hormone selected from the group that consists of kinetine, benziladenine (BA), Gibberelline (GA), and Zeatin. The method of Claim 5, wherein the culture medium contains a carbon source selected from the group that consists of saccharose (sucrose), fructose, and glucose. The method of Claim 3, wherein the oil is extracted from the culture medium with the individual cells that where multiplied from the individual cells generated from the explants derived from the A. Adding an organic solvent; B. Sonication; C. Centrifugation; D. Extraction of the superior face; and, E. Evaporation and drying of the solvent. A culture medium wherein, the culture medium comprises NH4NO3, CaNO3, CuSO, MnSO4, ZnSO4, H3BO3, KH2PO4, Na2MoO4, EDTA, FeSO4, CaCl2, CaCO3, NaC6H7O7 (sodium citrate), MgSO4, K2SO4, thiamine, glycine, inositol, nicotinic acid, pyridoxine, biotin, glutamine, naftalenacetic acid, zeatin, and a carbon source. The culture medium of Claim 11, wherein the culture medium contains cellulase, pectinase and hemicellulase. The culture medium of Claim 11, wherein the culture medium contains a salt selected from the group that consists of CaNO3 and KNO3; and/or a salt selected from the group that consists of CaCl2 and KC1. The culture medium of Claim 11, wherein the culture medium contains a hormone selected from the group that consists of indolacetic acid (IAA), naftalenacetic acid (NAA), and indolebutyric acid (IBA); and/or a hormone selected form the group that consists of kinetine, benziladenine (BA), Gibberelline (GA), and Zeatin. The culture medium of Claim 11, wherein the culture medium contains a carbon source selected from the group that consists of saccharose (sucrose), fructose, and glucose.BACKGROUND OF THE INVENTION
1. FIELD OF THE INVENTION
2. DESCRIPTION OF PRIOR ART
SUMMARY OF THE INVENTION
DETAILED DESCRIPTION OF THE INVENTION
NH4NO3 100 - 500 mg/L CaNO3, or KNO3 200 - 400 mg/L CuSO4 0.1 - 5 mg/L MnSO4 10 - 40 mg/L ZnSO4 20 - 50 mg/L H3BO3 10 - 20 mg/L KH2PO4 50 - 200 mg/L Na2MoO4 0.1 - 5 mg/L EDTA (ethylen-diamine-tetra-acetic acid) 10 - 50 mg/L FeSO4 10 - 50 mg/L CaCl2, or KCl 50 - 100 mg/L CaCO3 30 - 50 mg/L NaC6H7O7 (Sodium Citrate) 0.1 - 10 mg/L MgSO4 50 - 300 mg/L K2SO4 700 - 1500 mg/L thiamine 0.5 - 3 mg/L glycine 0.5 - 3 mg/L inositol 50 - 200 mg/L nicotinic acid 0.1 - 10 mg/L pyridoxine 0.1 - 10 mg/L biotin 0.1 - 10 mg/L glutamine 20 - 50 mg/L IAA, or NAA, or IBA 0.5 - 10 mg/L zeatin, or kinetine, or BA, or GA 0.5 - 10 mg/L saccharose, or glucose, or fructose 30000 - 100000 mg/L, and hemicellulase, and pectinase, and cellulase 3000 - 10000 mg/L NH4NO3 100 - 500 mg/L CaNO3, or KNO3 200 - 400 mg/L CuSO4 0.1 - 5 mg/L MnSO4 10 - 40 mg/L ZnSO4 20 - 50 mg/L H3BO3 10 - 20 mg/L KH2PO4 50 - 200 mg/L Na2MoO4 0.1 - 5 mg/L EDTA (ethylen-diamine-tetra-acetic acid) 10 - 50 mg/L FeSO4 10 - 50 mg/L CaCl2, or KCl 50 - 100 mg/L CaCO3 30 - 50 mg/L NaC6H7O7 (Sodium Citrate) 0.1 - 10 mg/L MgSO4 50 - 300 mg/L K2SO4 700 - 1500 mg/L thiamine 0.5 - 3 mg/L glycine 0.5 - 3 mg/L inositol 50 - 200 mg/L nicotinic acid 0.1 - 10 mg/L pyridoxine 0.1 - 10 mg/L biotin 0.1 - 10 mg/L glutamine 20 - 50 mg/L IAA, or NAA, or IBA 0.5 - 10 mg/L zeatin, or kinetine, or BA, or GA 0.5 - 10 mg/L saccharose, or glucose, or fructose 30000 - 100000 mg/L, and hemicellulase, and pectinase, and cellulase 3000 - 5000 mg/L EXAMPLES
NH4NO3 400 mg/L CaNO3 383 mg/L CuSO4 0.25 mg/L MnSO4 22.3 mg/L ZnSO4 8.6 mg/L H3BO3 6.2 mg/L KH2PO4 170 mg/L Na2MoO4 0.25 mg/L Na2EDTA (ethylen-diamine-tetra-acetic acid) 37.3 mg/L FeSO4 27.85 mg/L CaCl2 72.5 mg/L CaCO3 20 mg/L NaC6H7O7 (Sodium Citrate) 0.5 mg/L MgSO4 180.7 mg/L K2SO4 990 mg/L thiamine 1 mg/L glycine 2 mg/L mio-inositol 100 mg/L nicotinic acid 0.5 mg/L pyridoxine 0.5 mg/L biotin 1 mg/L glutamine 30 mg/L IAA 2 mg/L Kinetine 1 mg/L Saccharose 50000 mg/L, and pectinase, and cellulase 5000 mg/L Myristic 11.766 0.26 Nonanodioic acid 13.155 0.15 Palmitic acid 13.460 19.96 Palmitoleic 13.698 0.61 Estearic 14.994 8.64 Oleic 15.188 38.46 Linoleic 15.555 31.35 Eicosanoic 16.387 0.56 Palmitic 27.5±0.5 19.5±0.8 Estearic 10.5±0.4 6.8±0.6 Oleic 14.5±0.5 41.3±1.5 Linoleic 44.5±1.5 31.4±1.2 Linolenic 3.0±0.2 - Saturated 38 26.3 Unsaturated 62 72.7 Values are media ± standard deviation of duplicated determinations.