Abstract: Disclosed herein are media, kits, systems and methods for achieving micropropagation of bamboo on a commercially-relevant scale.

Abstract: The present invention relates to nutritive medium, medium supplement, media subgroup and buffer formulations. The present invention provides powder nutritive medium, medium supplement and medium subgroup formulations, e.g., cell culture medium supplements (including powdered sera such as powdered fetal bovine serum (FBS)), medium subgroup formulations and cell culture media comprising all of the necessary nutritive factors that facilitate the in vitro cultivation of cells. The invention further provides powder buffer formulations that produce particular ionic and pH conditions upon reconstitution with a solvent. The invention provides methods for production of media, media supplement, media subgroup and buffer formulations, and also provides kits and methods for cultivation of prokaryotic and eukaryotic cells, particularly bacterial cells, yeast cells, plant cells and animal cells (including human cells) using these dry powder nutritive media, media supplement, media subgroup and buffer formulations.

Abstract: The present invention relates to a method of Agrobaterium-based transformation of a plant comprising: generating a callus explant by, performing callus induction and maintenance in a plant in at least a first solution comprising less than about 100 mg/l of inositol; subjecting the callus explant to a cold treatment in a second solution comprising glutamine; and co-cultivating the callus explant with an Agrobacterium in a third solution comprising glutamine. The present invention also relates to a callus induction medium and/or callus maintenance medium, comprising: less than about 100 mg/l of inositol, and optionally at least one constituent selected from the group consisting of: an N6 salt or any plant culture media, proline, casein hydrolysate, a B-Vitamin, maltose, phytagel, 2,4-D, BAP and/or other plant growth regulators/hormones.

Abstract: The invention provides improved plant transformation methods. In particular the method provides increased transformation frequency, especially in recalcitrant plants. The method includes various transformation protocols for monocots, such as maize and sorghum, using a combination of media and light conditions to achieve increased efficiency of monocot transformation and increased callus initiation frequencies.

Abstract: The present disclosure relates, according to some embodiments, to citrus shoot regeneration compositions, methods, and systems. For example, citrus shoot regeneration compositions (e.g., culture media) may comprise one or more non-ionic surfactants. Methods may comprise preparing and using these compositions in some embodiments. Regeneration systems may comprise, in some embodiments, one or more of these compositions and/or one or more citrus explants.

Abstract: The present invention relates generally to nutritive medium, medium supplement, media subgroup and buffer formulations. Specifically, powdered nutritive medium, supplement, subgroup formulations, cell culture media comprising all of the necessary nutritive factors for in vitro cell cultivation, buffer formulations that produce particular ionic and pH conditions upon reconstitution with a solvent are provided. Particularly, methods of production of these media, supplement, subgroup, buffer formulations and kits, and methods for the cultivation of prokaryotic and eukaryotic cells using these dry powdered nutritive media, supplement, subgroup and buffer formulations are provided. Methods of producing sterile, powdered media or supplement (e.g., powdered FBS, powdered transferrin, powdered insulin, powdered organ extracts, powdered growth factors), media subgroup and buffer formulations by gamma irradiation are provided.

Abstract: The present invention provides methods for transforming monocot plants via a simple and rapid protocol, to obtain regenerated plants capable of being planted to soil in as little as 4-8 weeks. Associated cell culture media and growth conditions are also provided, as well as plants and plant parts obtained by the method. Further, a method for screening recalcitrant plant genotypes for transformability by the methods of the present invention is also provided. Further, a system for expanding priority development window for producing transgenic plants by the methods of the present invention is also provided.

Abstract: The present invention discloses a supercooling promoting agent comprising a tannin for producing practical water which does not freeze. As the tannin, a hydrolyzable tannin such as 2,3,6-tri-O-galloyl-?,?-D-hamamelose, 1,2,6-tri-O-galloyl-?-D-glucose, and a vitrification liquid, each of which contains the supercooling promoting agent are useful as a solution or the like for storing a biological material at low temperature.

Abstract: This invention provides methods whereby taxol, baccatin III, and other taxol-like compounds, or taxanes, can be produced in very high yield from all known Taxus species, e.g., brevifolia, canadensis, cuspidata, baccata, globosa, floridana, wallichiana, media and chinensis. Particular modifications of culture conditions (i.e., media composition and operating modes) have been discovered to enhance the yield of various taxanes from cell culture of all species of Taxus. Particularly preferred enhancement agents include silver ion or complex, jasmonic acid (especially the methyl ester), auxin-related growth regulators, and inhibitors of the phenylpropanoid pathway, such as 3,4-methylenedioxy-6-nitrocinnamic acid. These enhancement agents may be used alone or in combination with one another or other yield-enhancing conditions. While the yield of taxanes from plant cell culture of T.

Abstract: Auxotrophic Agrobacterium and methods employing auxotrophic Agrobacterium are provided. Auxotrophic Agrobacterium may be used in a variety of methods including biologically containing Agrobacterium comprising a transgene and transforming a plant cell without using an Agrobacterium counter-selective agent. Transforming maize immature embryos using an Agrobacterium auxotrophic for thymidine results in comparable transformation efficiency as transformation achieved using prototrophic Agrobacterium. Methods for producing an Agrobacterium thymidine auxotroph and using the auxotroph in transformation methods are disclosed. Transformed tissues and plants produced using methods of the present invention are also provided.

Abstract: Described herein are methods and media for facilitating somatic embryogenesis and for collecting, conditioning, and transferring the washed embryos onto a substrate and into an environment suitable for conditioning the embryos for a desired period of time so they become germination-competent for plant production. The described plant embryo cleaning apparatus and method are used for preparing multiple plant embryos for plant production. The apparatus and method can use a cleaning fluid source, a fluid-conditioning system, a fluid-delivery structure, a cleaning station, an outlet mechanism, a negative pressure source, and a controller.

Abstract: The present invention relates generally to nutritive medium, medium supplement, media subgroup and buffer formulations. Specifically, powdered nutritive medium, supplement, subgroup formulations, cell culture media comprising all of the necessary nutritive factors for in vitro cell cultivation, buffer formulations that produce particular ionic and pH conditions upon reconstitution with a solvent are provided. Particularly, methods of production of these media, supplement, subgroup, buffer formulations and kits, and methods for the cultivation of prokaryotic and eukaryotic cells using these dry powdered nutritive media, supplement, subgroup and buffer formulations are provided. Methods of producing sterile, powdered media or supplement (e.g., powdered FBS, powdered transferrin, powdered insulin, powdered organ extracts, powdered growth factors), media subgroup and buffer formulations by gamma irradiation are provided.

Abstract: Compositions and methods are provided for the efficient transformation of a dicot plant. More particularly, compositions and methods of the present invention find use in agriculture for Agrobacterium-mediated transformation of a dicotyledonous plant. The compositions include cultivation media comprising high concentrations of sucrose and glucose. The cultivation media find use in methods directed to Agrobacterium-mediated transformation of a dicot plant with a gene of interest. In this manner, any gene of interest can be introduced into a dicot plant with high transformation efficiency and reduced tissue necrosis.

Abstract: Described herein are methods and media for facilitating somatic embryogenesis and for collecting, conditioning, and transferring the washed embryos onto a substrate and into an environment suitable for conditioning the embryos for a desired period of time so they become germination-competent for plant production. The described plant embryo cleaning apparatus and method are used for preparing multiple plant embryos for plant production. The apparatus and method can use a cleaning fluid source, a fluid-conditioning system, a fluid-delivery structure, a cleaning station, an outlet mechanism, a negative pressure source, and a controller.

Abstract: The subject invention provides simple and consistent methods to break suspension cell aggregates to single cells with intact primary cell walls. The subject invention relates in part to cell separation of suspension cell aggregates cultured in medium containing pectin-degrading enzymes or tubulin de-polymerizing compounds including colchicine. The subject invention also relates to novel uses of compounds for such purposes. Another aspect of the subject invention relates to transformation of the subject, isolated cells. Such processes simplify and integrate single-cell-based transformation and selection processes into transgenic and transplastomic event-generation work processes. The subject invention also removes technical constraints and produces marker-free and uniformly expressing transgenic lines in a high throughput fashion to support various needs of animal health, biopharma, and trait and crop protection platforms.

Abstract: Improved methods for the regeneration of transgenic plants using apical dominance inhibitors are disclosed. Use of dikegulac to induce multiple shoot formation in culture is particularly disclosed.

Abstract: The present invention is generally in the field of continuous and high-cell-density cultivation in laboratory- or large-scale liquid shaken cultures. More particularly it relates to a method of enzyme-based fed-batch (EnBase) for liquid microbial prokaryotic or eukaryotic cell cultivation having the possibility to manipulate the growth rate of the cultured organisms by a controlled enzymatic release of the growth-limiting substrate-monomer from substrate-polymers or substrate-oligomers.

Abstract: Particular aspects provide compositions comprising an electrokinetically altered oxygenated aqueous fluid, wherein the oxygen in the fluid is present in an amount of at least 25 ppm. In certain aspects, the electrokinetically altered oxygenated aqueous fluid comprises electrokinetically modified or charged oxygen species present in an amount of at least 0.5 ppm. In certain aspects the electrokinetically altered oxygenated aqueous fluid comprises solvated electrons stabilized by molecular oxygen, and wherein the solvated electrons present in an amount of at least 0.01 ppm. In certain aspects, the fluid facilitates oxidation of pyrogallol to purpurogallin in the presence of horseradish peroxidase enzyme (HRP) in an amount above that afforded by a control pressure pot generated or fine-bubble generated aqueous fluid having an equivalent dissolved oxygen level, and wherein there is no hydrogen peroxide, or less than 0.1 ppm of hydrogen peroxide present in the electrokinetic oxygen-enriched aqueous fluid.

Abstract: The present invention provides a method of transforming common ice plant by gene transfer using a microorganism belonging to the genus Agrobacterium and a method of producing a transformed common ice plant. The present invention also provides a stable transformed common ice plant.

Abstract: The present description concerns methods for regeneration of whole plant from the explants obtained from the Abelmoschus species preferably A. esculentus. In addition the present description also concerns methods for transforming okra plant, plant cells and tissues either with the use of recombinant Agrobacterium strain or by bombarding the explants with tungsten or gold particles coated with DNA sequences of interest. An efficient method to isolate embryos from imbibed seeds of okra is also described which enables the use of young meristematic cells of plumule tip for efficient regeneration and transformation of okra plants. Further, transformed okra plants, plant cells and tissues for improved agronomic/non agronomic traits and insect resistance are produced either by using marker based or marker free systems.

Abstract: Cell culture media, concentrated media and feeds, methods of manufacturing cell culture media and feeds, and methods of culturing cells are provided. One or more small peptides, including dipeptides are added to the cell culture media to provide improved stability and improved conditions for culturing cells.

Abstract: The present invention discloses rucola plants, including an E. sativa plant, with cytoplasmic inherited male sterility (CMS) for hybrid breeding purposes. The present invention includes plants that comprise CMS-cytoplasm from cauliflower (B. oleracea var. botrytis) transferred to E. sativa by a wide interspecific cross.

Abstract: The present invention relates to excision of explant material comprising meristematic tissue from cotton seeds. Methods for tissue preparation, storage, transformation, and selection or identification of transformed plants are disclosed, as are transformable meristem tissues and plants produced by such methods, and apparati for tissue preparation.

Abstract: The present invention provides a method for improving the preparation and use of growth media by the uses of specific pellet formulations, which especially are tablets of different sizes, which contain the growth medium or parts thereof and are sterilized with the standard methods of pharmaceutical technology. Specifically these pellet formulations are applied to control a cell culture in a way that the adaptation phase is shorter or that the growth is controlled by a release of certain components at a certain time and in a certain concentration during the process, and nutrients (e.g., nitrogen) can be packed into the cultivation vessel in amounts sufficient for high cell densities without the risk of intoxication of the organism.

Abstract: The present invention relates to methods and compositions for transforming soybean, corn, cotton, or canola explants using spectinomycin as a selective agent for transformation of the explants. The method may further comprise treatment of the explants with cytokinin during the transformation and regeneration process.

Abstract: The present invention relates to a composition for preventing or treating cancer, which contains, as an active ingredient, a Panax ginseng cambium-derived cell line including wild ginseng or ginseng; a lysate thereof; an extract thereof; or a culture medium thereof. The cell line according to the present invention, a lysate thereof, an extract thereof and a culture medium thereof are derived from a natural and have minimized side effects compared to the conventional therapeutic drugs, and thus are safe for the human body. Also, they are involved directly in the growth of cancer to induce cancer cell death effectively, and show anticancer activity of inhibiting or reducing the formation of tumor or the growth of tumor, Accordingly, the cell line, the lysate thereof, the extract thereof and the culture medium thereof are useful for the prevention, treatment and alleviation of cancer.

Abstract: A method for producing transgenic plants, including treating a target tissue using plasmolyzing media (PM) which contains 4% to 10% of sucrose and 100 ?M to 300 ?M of Acetosyringone (AS) and gold particles. The target tissue is infected by a bacterial suspension using a suitable strain and a suitable transformation vector. A PM containing 4% to 10% sucrose and 100 ?M to 300 ?M AS is treated for a period between 1 to 3 days. Cultivation is performed in a cultivation media in a dark condition at a temperature between 25° C. to 30° C. A non-selection media with an antibiotic is introduced. A selection media containing an active ingredient phosphinothricin (PPT) is introduced in a light condition at a temperature of between 25° C. to 30° C. in a sub culture for a period of between 3 weeks to 1 month. The putative transformant is regenerated and the number of copies of the transgenes is analyzed.

Abstract: The subject invention provides simple and consistent methods to break suspension cell aggregates to single cells with intact primary cell walls. The subject invention relates in part to cell separation of suspension cell aggregates cultured in medium containing pectin-degrading enzymes or tubulin de-polymerizing compounds including colchicine. The subject invention also relates to novel uses of compounds for such purposes. Another aspect of the subject invention relates to transformation of the subject, isolated cells. Such processes simplify and integrate single-cell-based transformation and selection processes into transgenic and transplastomic event-generation work processes. The subject invention also removes technical constraints and produces marker-free and uniformly expressing transgenic lines in a high throughput fashion to support various needs of animal health, biopharma, and trait and crop protection platforms.

Abstract: The present invention provides methods for regrowth of conifer embryogenic cells. The methods comprise the steps of (a) contacting cryopreserved conifer embryogenic cells with a liquid transition medium, and (b) culturing the contacted conifer embryogenic cells in a regrowth medium to generate regrowth of the conifer embryogenic cells. Generally, the cryopreserved conifer embryogenic cells are contacted with the liquid transition medium for less than about 24 hours, such as for about one hour.

Abstract: The present invention relates to improved methods for the incorporation of DNA into the genome of a Zea mays plant by means of Agrobacterium-mediated transformation. Preferred is the use of the Zea may lines deposited with American Type Culture Collection under the Patent Deposit Designation PTA-6170 and PTA-6171.

Abstract: The present invention is directed generally to metal binding compounds which may be added to cell culture media to replace factors required for cultivation of the cells (e.g. transferrin) which are of animal or human origin. More specifically, the invention is directed to metal binding compounds or complexes thereof comprising one or more transition element cations (such as ferrous or ferric ions), which are added to cell and tissue culture medium compositions. The metal binding compounds may be added to the media alone or may be first complexed with a transition metal ion. The invention is also directed to methods of use of such compositions, including, for example, methods for the cultivation of eukaryotic cells, particularly animal cells, in vitro. The invention also relates to compositions comprising such culture media and one or more cells, and to kits comprising one or more of the above-described compositions.

Abstract: 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.

Abstract: 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.

Abstract: A novel maize variety designated PHHEN and seed, plants and plant parts thereof. Methods for producing a maize plant that comprise crossing maize variety PHHEN with another maize plant. Methods for producing a maize plant containing in its genetic material one or more traits introgressed into PHHEN through backcross conversion and/or transformation, and to the maize seed, plant and plant part produced thereby. Hybrid maize seed, plant or plant part produced by crossing the variety PHHEN or a trait conversion of PHHEN with another maize variety. Inbred maize varieties derived from maize variety PHHEN, methods for producing other inbred maize varieties derived from maize variety PHHEN and the inbred maize varieties and their parts derived by the use of those methods.

Abstract: The present invention relates to a method for the production of somatic embryos from poinsettia (Euphorbia pulcherrima) plant tissue. Furthermore, the present invention discloses subject matter that relates to poinsettia plants, particularly to non-chimeric poinsettia plants, and to a method of producing the same.

Abstract: The present invention is in the field of plant breeding and genetics, particularly as it pertains to the genus, Glycine. More specifically, the invention relates to a method for screening soybean plants containing one or more quantitative trait loci for disease resistance, species of Glycine having such loci and methods for breeding for and screening of Glycine with such loci. The invention further relates to the use of exotic germplasm in a breeding program.

Abstract: A preservation solution for preserving a biological material at a low temperature including one or more polyphenols, and a method for preserving a biological material are provided. The method includes adding the preservation solution to a biological material, cooling the biological material and storing it under appropriate storing conditions. The method can be used for hypothermic preservation or for cryopreservation, including freezing and lyophilization, of any biological material, including cells selected from RBC, WBC, MNC, UCB, HSC and bacteria. A method is also provided for freezing RBCs such that upon thawing, the RBCs include less than 2% free hemoglobin.

Abstract: Acceleration of botryococcenoids and growth by concomitant provision of appropriate light, minerals, and assimilable carbon. Specifically, methods, compositions and systems for the in vitro growth of hydrocarbons in photosynthetic organisms while maintaining a biologically exclusive monocultural environment, as for example, from Botryococcus species, is disclosed. Niche-nutrients can include about 200 ppm to about 3% nitrogen, and about 100 ppm to about 15% P2O5, and about 100 ppm to about 3.5% K2O. In certain embodiments, the present invention relates to the growth of the Chlorophyta such as Botryococcus sp. in a nutrient medium that includes up to 15% phosphates, at least 3 ppm soluble iron, and up to about 70 ppm soluble zinc. Also disclosed is a substantially pure culture of Botryococcus braunii var. Showa, strain Ninsei, having the ATCC Accession No. PTA-7441, its parts, and hydrocarbons produced therefrom.

Abstract: The present invention provides a convenient method for producing a transformed plant by expressing a hormone gene positioned within a plasmid backbone that also carries a P-DNA or T-DNA to obtain backbone-free transformed plants.

Abstract: In one aspect, a method is provided for producing an improved nutritive medium comprising an adsorbent material for culturing plant embryos.

Abstract: A method of mass producing potato seedlings, involves collecting growing points of seed potatoes and culturing the growing points in a liquid or solid medium; introducing in vitro plantlets obtained from the culture of the growing points to solid culture; and removing the in vitro plantlets from the solid culture, and planting through stem cutting and acclimatizing the in vitro plantlets in deep flow culture, in which a nutrient solution is circulating. Upon planting in hydroponic facilities, such potato seedlings have high adaptability to the external environment and thus rapidly, uniformly generate roots in a short time. The rapid root anchoring prevents planted seedlings from withering, leading to death, growing poorly, and the like. The direct planting of in vitro plantlets through stem cutting without a separate acclimatization process shortens the overall production period of potato seedlings by omitting the acclimatization process.

Abstract: The present invention provides methods for high efficiency plant transformation via Agrobacterium-mediated T-DNA conjugation to suspension-cultured cells or calli. The methods described herein employ membranes or filters as porous solid support for the co-culture of T-DNA donor and recipient.

Abstract: The present invention provides a method for producing a true-to-type clone of an Aloe b?rb?densis mother plant by selecting an Aloe b?rb?densis mother plant; isolating a meristematic explant from the plant; culturing the meristematic explant in initiation medium to generate shoots, where the media lacks hormones; culturing the shoots in proliferation and elongation medium to generate elongated shoots, where the proliferation and elongation media comprises benzyl adenine (BA) and indole butyric acid (IBA); culturing the elongated shoots in rooting medium to generate plantlets, where the media lacks hormones; and culturing the plantlets to product a true-to-type clone of the Aloe b?rb?densis mother plant.

Abstract: The invention relates to methods for Rhizobia-mediated genetic transformation of plant cells, including soybean, canola, corn, and cotton cells. These include both VirD2-dependent and VirD2-independent methods. Bacterial species utilized include strains of Rhizobium sp., Sinorhizobium sp., and Mesorhizobium sp. Vectors for use in such transformation are also disclosed.

Abstract: The present invention describes a method for increasing the survival of the bacteria of Rhizobium genus, comprising the steps of: making the bacteria to grow in a chemically defined medium; keeping the bacteria in growth stationary phase for a proper period of time; exposing the bacteria to effective quantities of indole-3-acetic acid (IAA). Within the invention scope there is an alternative method to increase the survival of the bacteria of the Rhizobium genus by means of genetic engineering comprising the steps of: making a recombinant vector codifying enzymes able to produce IAA to express in effective way in said bacteria; making the bacteria to grow in chemically defined culture medium; keeping the bacteria in growth stationary phase for a proper period of time.

Abstract: A method of sowing a somatic plant embryo or germinant of a conifer species to facilitate subsequent development of the embryo or germinant into an autotrophic seedling. The method involves the following steps carried out ex vitro in non-sterile conditions: providing a nutrient medium comprising particles of a solid component present within a flowable or semi-solid component containing water and a carbohydrate nutrient for the embryo or germinant, dispensing a quantity of the nutrient medium onto a surface of a porous solid growth substrate for the somatic plant embryo or germinant, to and contacting the plant embryo or germinant with the nutrient medium. The nutrient medium has a fluidity such that at least some of the flowable or semi-solid component containing the carbohydrate nutrient remains in contact with the embryo or germinant at least until the embryo or germinant establishes vigorous growth under environmental conditions effective for such growth.

Abstract: The present invention provides an in vitro flavonoid-rich rhizome tissue of Neomarica gracilis, which is obtained from a tissue culture preparation of an N. gracilis tissue capable of proliferating, such as a root, a leaf, a basal portion of a leaf, and/or a rhizome. The in vitro flavonoid-rich rhizome tissue of N. gracilis contains tectorigenin, which is distinctively different from the naturally grown rhizome of N. gracilis which contains no tectorigenin. The present invention further provides a method for cultivating the in vitro flavonoid-rich rhizome tissue, a method for extracting the tectorigenin from the flavonoid-rich rhizome tissue, and quantitative methods for determining the amount of tectorigenin in the in vitro flavonoid-rich rhizome tissue.

Abstract: A plant culture medium composition for modulating plant transformation events, comprising a plant culture medium and an effective amount of at least one compound having a chloride component intermixed thereinto. In one embodiment, the at least one chloride-containing compound is selected from the group comprising: NaCl, MgCl2, and KCl. Another embodiment relates to a method for modulating the frequency of plant transformation events. The method comprises the steps of providing a plant culture medium composition and contacting at least one plant with the plant culture medium composition. At least one cell from the at least one plant is transformed with a nucleic acid of interest. The presence of at least one transformation event is detected and quantified. The frequency of quantified transformation events is compared with a suitable control. Changes in quantified transformations events compared to the control are indicative of changes in the frequency of plant transformation events.

Abstract: The disclosed invention relates to the use of esters of unsaturated, physiologically active fatty acids as nutrient media for cell cultivation and, more particularly, as a substitute for foetal bovine serum. In one aspect, the esters comprise more than 50 mol-% of physiologically active fatty acids containing 16 to 24 carbon atoms and 2 to 5 double bonds as the acid component and a lower C1-4 alcohol, preferably ethanol, or a sterol as ester component. In another aspect, the esters comprise a transesterification product of natural or synthetic oils or a mixture of such oils having greater than 50 mol-% of unsaturated, physiologically active fatty acids, based on the acyl group, and a lower C1-4 alcohol or a sterol. In a further aspect, the esters are used together with sterols, phospholipids and/or vegetable proteins or are liposomally encapsulated.

Abstract: The present invention provides a method for Agrobacterium-mediated gene introduction into a plant material, comprising: 1) treating the plant material, and then 2) infecting the plant material with an Agrobacterium, characterized in that a medium enriched in a metal salt containing copper ion is used in step 1) and/or 2). The present invention also provides a process for preparing a transformed plant characterized in that the gene introduction method of the present invention is used.