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    • 专利摘要:

    公开号:SE535010C2
    申请号:SE1051008
    申请日:2010-09-29
    公开日:2012-03-13
    发明作者:Jeffrey E Hartle；William C Carlson
    申请人:Weyerhaeuser Nr Co；
    IPC主号:
    专利说明:

    In one embodiment, the shot restriction further includes an adsorbent material in the cavity. In one embodiment, the adsorbent material in the cavity is charcoal. In one embodiment, the adsorbent material in the cavity is a nutrient-treated charcoal. In another aspect, the present invention provides methods for improving the germination of a plant embryo based on a manufactured seed. In one embodiment, the method comprises the steps of (a) assembling a manufactured seed including a seed coat and a shoot restriction, the shoot restriction comprising a cavity; (b) adding nutrient medium including an adsorbent material to the seed coat, the adsorbent material being present in the medium at a concentration of from about 30 g / l to about 100 g / l; (c) placing a plant embryo in the cavity of the shoot restriction; and (d) culturing the produced seed under conditions suitable for germination of the plant embryo.
    In one embodiment, the method comprises the steps of (a) assembling a manufactured seed including a seed coat and a constraint, the constraint comprising a cavity; (b) adding nutrient medium including an adsorbent material to the seed coat, the adsorbent material presenting the medium in a concentration of from about 30 g / l to about 100 g / l; (c) placing a plant embryo in the cavity of the restriction; (d) adding an adsorbent material to the cavity; and (e) culturing the produced seed under conditions suitable for germination of the plant embryo.
    It is to be understood that the order of steps of the process may change without departing from the spirit of the invention. An adsorbent material can, for example, be placed in the cavity of the restriction before the embryo is placed in the restriction.
    Description of the Drawings The above aspects and many of the attendant advantages of the present invention will become more apparent and more readily understood by reference to the following description taken in conjunction with the accompanying drawings. Fig. 1 shows in cross-sectional view an example of a manufactured seed including an embryo for use in the methods of the present invention. 10 15 20 25 30 535 CVIÜ 3 FIGURE 2 shows a diagram of the degrees of germination of embryos placed in a manufactured seed in relation to increasing amounts of charcoal in the nutrient medium. Observations were made 19 days after sowing.
    FIGURE 3 shows a diagram of the degrees of germination of embryos placed in a manufactured seed in relation to increasing amounts of charcoal in the nutrient medium. Observations were made 26 days after sowing.
    FIGURE 4 shows a diagram of organ lengths for embryos placed in a manufactured seed in relation to increasing amounts of charcoal in the nutrient medium. Observations were made 35 days after sowing.
    FIGURE 5 shows a diagram of the presence of embryos placed in a manufactured seed in relation to increasing amounts of charcoal in the food.
    Observations were made 35 days after sowing.
    Detailed Description Unless otherwise specified, all terms used herein have the same meaning as they would be apparent to those skilled in the art to the present invention.
    Unless otherwise stated, all concentration values are expressed as a percentage by weight per unit volume.
    In one aspect, the present invention provides a manufactured seed comprising (a) a seed coat; (b) a nutrient medium comprising an adsorbent material, wherein the adsorbent material is present in the medium in a concentration of from about 30 g / l to about 100 g / l (eg from about 50 g / l to about 100 g / l, from about 60 g / l to about 100 g / l, or from about 75 g / l to about 100 g / l); and (c) a shot restriction, the shot restriction including a cavity. In one embodiment, the seed produced also includes a plant embryo.
    FIGURE 1 shows in cross-sectional view an example of a manufactured seed 20 including a plant embryo 42 placed therein. As shown in FIGURE 1, the embryo 42 is located within a cavity 34, is in functional contact with a nutrient medium 26 and is suitably enclosed therein by means of a living end closure 43. It will be appreciated that Figure 1 provides a representative embodiment of a manufactured seed 20, but the method according to the invention is not limited to the particular embodiment of the manufactured seed shown in FIGURE 1. In the exemplary embodiment shown in FIGURE 1, the manufactured seed 20 includes the manufactured seed 20. a seed coat 24, a nutrient medium 26, a bottom closure 28 and an optional shoot restriction 22.
    The term "seed coat" as used herein refers to a structure analogous to that of a natural seed coat which protects the plant embryo and other internal structures of the seed produced from mechanical damage, dehydration, attack by microbes, fungi, insects, nematodes, birds and other pathogens, herbivores and pests, to name a few. The seed coat 24 may be made of a variety of materials including, but not limited to, cellulosic materials, glass, plastic, mouldable plastic, cured polymeric resins, paraffin, waxes, varnishes, and combinations thereof, such as a wax impregnated paper. The materials from which the seed coat is made are generally non-toxic and provide a degree of rigidity. The seed coat may be biodegradable, although the seed coat usually remains intact and resistant to penetration of plant pathogens until after the emergence of the germinating embryo. The seed coat may be formed from a part of a tubular material. The seed coat can be a cut straw made of fibrous material, such as paper. The suction pipe parts can be pretreated in a suitable coating material, such as wax. Alternatively, the seed coat may be formed on the basis of a tubular part of a biodegradable plastic material. One such material is polylactic acid ("PLA"), sold by NAT-UR in Los Angeles, California. Another suitable material is a polycaprolactone ("PCL") blend, such as CAPA 6800 (Perstorp Polyols lnc., Toledo, Oklahoma 43612) with or without a plasticizer in the form of 1% Tegomer H Sl644O (Degussa Goldschmidt Chemical Corp., 914 East Randolph Road, Hopewell, Virginia 23860) Such biodegradable plastic tubes require or do not require a wax coating, as such tubes Additives, such as antibiotics and plant growth regulators, can be added to the seed coat, for example, by incorporating therein to provide one or more layers of the seed coat or by coating or otherwise treating the layer or layers with the additive by conventional means.
    With reference to the manufactured seeds and methods of the invention, the nutrient medium 26 is in functional contact with the plant embryo located in the manufactured seed 20. The term "nutrient medium" as used herein refers to a source of nutrients such as vitamins, minerals, carbon and 15 20 25 30 535 010 5 energy sources, and other beneficial compounds used by the embryo during germination. The nutrient medium 26 is thus analogous to the gametophyte of a natural seed. A nutrient medium 26 according to the invention may include a substance which causes the medium to become semi-solid or has a solidified consistency under normal ambient conditions. Usually the nutrient medium 26 is in the form of a hydrated gel. A "gel" is a substance which is prepared as a colloidal solution and which forms or can be made to form a semi-solid material. Such a conversion of a surface gel solution to a semi-solid material is referred to herein as "curing" or "solidification" of the gel. The term "hydrated gel" refers to an aqueous gel, such gels being prepared by first dissolving in water ( where the water acts as a solvent or "continuous phase") of a hydrophilic polymeric substance (which acts as a solute or "dispersed phase"), which on curing is combined with the continuous phase to form the semi-solid material, thus the water becomes homogeneous associated with the dissolved molecules without any significant separation of the continuous phase from the dispersed phase.However, water molecules can be freely removed from a cured hydrated gel, such as by evaporation or aspiration of a germinating embryo.In cured state, these gels have the property of resilient solids, such as a gelatin mass, where the resilience gradually decreases and the gel becomes more "solid" to the touch when the relative amount of v eighteen in the gel decreases.
    In addition to being water-soluble, suitable solutes for the gel are neither cytotoxic nor substantially phytotoxic. As used herein, the term "substantially non-phytotoxic" is a substance which does not significantly interfere with normal plant development, such as by killing a significant number of plant cells, substantially altering cellular differentiation or maturation, causing mutations, destroying a significant number of cell membranes or significantly disrupt cellular metabolism or significantly disrupt any other process.
    Suitable solutes for the gel include, but are not limited to, the following: sodium alginate, agar, agarose, amylose, pectin, dextran, gelatin, starch, amylopectin, modified celluloses, such as methylcellulose and hydroxyethylcellulose, and polyacrylamide. Other hydrolyzable solutes in the gel can also be used as long as they have similar hydration and gelling properties and lack toxicity.
    The gels are usually prepared by dissolving a substance to be dissolved in the gel, usually in a fine particulate form, in water to form a gel solution. Depending on the particular substance to be dissolved in the gel, heating is usually necessary, sometimes even until boiling, before the substance to be dissolved in the gel dissolves. Subsequent cooling causes many gel solutions to "solidify" or "harden" reversibly (gel). Examples include gelatin, agar and agarose. Such solutes in the gel are termed "reversible", since reheating a cured gel regenerates the gel solution. Solutions of other solutes in the gel require a "complexing" substance, which serves to chemically cure the gel by crosslinking dissolved molecules in the gel. Sodium alginate is cured, for example, by the addition of calcium nitrate (Ca (NO3) 2) or salts of other divalent ions to the gel solution, such as, but not limited to, calcium, barium, lead, copper, strontium, cadmium, zinc, nickel, cobalt, magnesium and even. Many of the solutes in the gel that require complexing agents are irreversibly cured, with reheating not re-establishing the gel solution.
    The concentration of the solute in the gel varies depending on the particular solvent of the gel. A useful concentration of sodium alginate is, for example, in a range of about 0.5% to about 2.5% (w / v), preferably about 0.9% to 1.5% (w / v). A useful concentration of agar is in the range of about 0.8% to about 2.5% (w / v), preferably about 1.8% (w / v). Gels cured by complex formation generally require less solute for the gel to form a satisfactory gel compared to “reversible” gels.
    The nutrient medium 26 usually includes one or more carbon sources, an adsorbent material, vitamins and minerals. Suitable carbon sources include, but are not limited to, monosaccharides, disaccharides, and / or starches. Suitable adsorbent materials include, but are not limited to, charcoal, polyvinylpolypyrrolidone, and silica gels. The nutrient medium 26 may also include amino acids and a smoke suspension. Suitable amino acids include amino acids that are usually incorporated into proteins, as well as amino acids that are not usually incorporated into proteins, such as arginine succinate, citrulline, canavanine, ornithine and D-stereoisomers. A suitable smoke suspension contains one or more compounds formed by the process of burning organic material, such as wood or other cellulosic material. Solutions containing these by-products from the combustion of organic materials can be formed by burning the organic material, washing the charred material with water and collecting the water. The solutions can also be obtained by heating the organic material and condensation and dilution of volatile substances which are released during such heating. Certain types of smoke suspensions can be purchased from commercial suppliers, such as Wright's Concentrated Hickory Seasoning Liquid Smoke (B&G Foods, Inc. Roseland, New Jersey 07068). Smoke suspension can be incorporated into the nutrient medium 26 in any of a number of forms. The smoke suspension can, for example, be incorporated as an aerosol, a powder or as an activated clay. An example of the concentration of the fl liquid smoke suspension, if any, is, according to Wright's Concentrated Hickory Seasoning Liquid Smoke, between 0.0001 ml and 1 ml of smoke suspension per liter of medium. The nutrient medium 26 may also include one or more compounds involved in nitrogen metabolism, such as urea or polyamines.
    The nutrient medium 26 includes oxygen carriers to enhance both the absorption of oxygen and the retention of oxygen for the nutrient medium 26, which enables the medium to maintain an oxygen concentration higher than that which would otherwise be present in the medium simply by the absorption of oxygen from the atmosphere. Examples of oxygen carriers include perohydrocarbons, such as FC-77 (3M Corporation, St. Paul, Minnesota), emulsified with a surfactant, such as Pluronic F-68, available from BASF Corp., Parsippany, New Jersey. Examples of oxygen carriers are described in U.S. Patent Application No. 5,564,224 (e.g., column 9, line 44, to column 11, line 67), which is hereby incorporated by reference.
    The nutrient medium 26 may also contain hormones. Suitable hormones include, but are not limited to, abscisic acid, cytokinins, auxins, and gibberellins. Abscisic acid is a sesquiterpenoid plant hormone that is implicated in a number of plant physiological processes (see, e.g., Milborrow, J. Exp. Botany 52: 1145-1164 (2001); Leung & Giraudat Ann. Rev. Plant Physiol. Plant Mol. 10 15 20 25 30 535 CHÜ 8 Biol. 49: 199-123 (1998)). Auxins are plant growth hormones that promote cell division and growth. Examples of auxins for use in the germination medium include, but are not limited to, 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, indole-3-butyric acid, naphthalene acetic acid and hydroxycinnamic acid. Cytokinins are plant growth hormones that affect the organization of cells during division. Examples of cytokinins for use in the germination medium include, but are not limited to, for example, 6-benzylaminopurine, 6-furfurylaminopurine, dihydrogenzeatin, zeatin, kinetin and zeatin riboside. Gibberellins are a class of diterpenoid growth hormones (see, for example, Krishnamoorthy Gibberellins and Plant Growth, John Wiley & Sons, (1975)). Representative examples of gibberellins useful in the practice of the present invention include gibberellic acid, gibberellin 3, gibberellin 4 and gibberellin 7. An example of a useful mixture of gibberellins is a mixture of gibberellin 4 and gibberellin 7 (designated gibberellin 7). 7), such as the 4/7 gibberellin sold by Abbott Laboratories, Chicago, Illinois.
    The nutrient medium 26 may also include antimicrobial agents. Suitable antimicrobials are available from Sigma-Aldrich, St. Louis, Missouri, and sold as Product No. A5955. Antimicrobial substances can, for example, be used in a concentration of 1 ml / l.
    When abscicic acid is present in the nutrient medium 26, it is usually used in a concentration of in the range of from about 1 mg / L to about 200 mg / L. When present in the nutrient medium 26, the concentration of gibberellin (s) is usually between about 0.1 mg / L and about 500 mg / L. Auxins can be used, for example, in a concentration of from 0.1 mg / l to 200 mg / l. For example, cytokinins can be used in a concentration of from 0.1 mg / L to 100 mg / L.
    Examples of nutrient media are described in U.S. Patent No. 5,687,504 (e.g., column 8, line 63 to column 9, line 41) and U.S. Patent Publication No. 2003/0167684, which is hereby incorporated by reference. A representative nutrient medium 26 is KE64-50, the composition of which is described in Table 1 below.
    In one embodiment of the invention, the concentration of the adsorbent material in the nutrient medium 26 is from about 30 g / l to about 100 g / l. In another embodiment, the concentration of the adsorbent material in the nutrient medium is from about 50 g / l to about 100 g / l. In another embodiment, the concentration of the adsorbent material in the nutrient medium 26 is from about 60 g / l to about 100 g / l. In another embodiment, the concentration of the adsorbent material in the nutrient medium 26 is from about 75 g / l to about 100 g / l. In one embodiment, the adsorbent material is charcoal. In one embodiment, the adsorbent material is nutrient-treated charcoal. The term "nutrient-treated" charcoal as used herein refers to charcoal which has been treated with a medium containing a variety of nutrients, such as a carbon source, vitamins, minerals and amino acids, the charcoal absorbing and conserving nutrients from the medium. used for the preparation of nutrient-treated charcoal is the medium KE64- 50. A representative process for the production of nutrient-treated charcoal is described in Example 2.
    The shoot limitation 22 of a manufactured seed is suitably made from a porous material having a hardness strong enough to withstand puncture or fracture due to a germinating embryo, such as a ceramic material or a porcelain material, and includes an end closure portion 30 and a leaf blade restriction portion 32. The restriction portion 32 has an inner surface for contacting and enclosing at least the shoot end of a plant embryo and resists penetration of the shoot end during germination. The shoot restriction prevents the shoot end of the embryo, such as the heart leaves, from growing into and enclosing it in the nutrient medium (also called nutrient medium).
    The heart blade restriction portion 32 is suitably integrally or uniformly formed together with the end closure portion 30. The shot restriction 22 also includes a longitudinally extending cavity 34 extending through the end closure portion 30 and partially through one end of the heart leaf restriction portion 32. The open end is the open end 34. known as a heart leaf restriction opening 36. The cavity 34 is sized to receive a plant embryo 42 therein. As shown in Figure 1, the shot restriction 22 includes a plurality of pores 27, the pores 27 providing the nutrient medium 26 with access to the inside of the cavity 34 including the embryo 42 and therefore allowing the nutrient medium 26 to functionally contact the embryo 42 under conditions sufficient to give a conditioned embryo, as described here. 10 15 20 25 30 535 010 10 The restriction is porous to give the embryo access to water, nutrients and oxygen. The shot restriction may be made of any suitable material which includes, but is not limited to, glassy material, metal material, elastomeric material, ceramic material, clay material, gypsum material, cement material, starch material, putty-like material, synthetic polymeric material, natural polymeric materials and adhesives. Examples of shot restrictions are described in U.S. Patent No. 5,687,504 (e.g., column 3, line 61 to column 4, line 13; column 18, line 7, to column 22, line 2), which is hereby incorporated by reference.
    All or only part of the plant embryo 42 may be inserted into the shoot restriction 22. Usually, at least the shoot end of the embryo is inserted into the shoot restriction 22. The surface for nutrient uptake of a produced seed 20 is limited to the surface of the plant embryo 42 which is in direct contact with the inner surface. of the shoot restriction 22. During germination of plant embryos, the heart leaves have been shown to be the primary organs for nutrient uptake (Brown & Gifford, Plant Physiol. 33: 57-64 (1958)).
    Either the inner surface of the shoot restriction 22 or the plant embryo 42, or both, can be contacted with a hydrated gel, either before or after insertion of the plant embryo 42 into the shoot restriction 22. Examples of embodiments of hydrated gels are as described above for the nutrient medium 26. The hydrated gel may comprise only solutes for the gel and water, or it may include plant nutrients and other substances, as described for the nutrient medium 26. The inner surface of the shoot restriction may be contacted with a hydrated gel solution which is cured to form a hydrated gel. A cavity 34 may then be provided in the hydrated gel in the shoot restriction 22, and the plant embryo 42 may be inserted into the cavity 34 in the hydrated gel in the shoot restriction 22. In addition, or alternatively, at least a portion of the plant embryo 42 (such as the heart leaves) may be contacted. a hydrated gel solution which is cured to form a hydrated gel prior to insertion of the plant embryo 42 into the shoot boundary 22.
    As further shown in FIGURE 1, in one embodiment of the invention, the adsorbent material 80 surrounds the embryo 42 in the cavity 34, either completely or partially, and increases the surface area of the embryo 42, which is in functional contact with nutrient medium 26, which provides a number of pathways for the nutrients from nutrient medium 26 to reach the embryo 42. Suitable adsorbent materials include activated charcoal, Dowex resins, zeolites, alumina, clay, diatomite, silica gel and kieselguhr. During the assembly of the manufactured seed 20, the adsorbent material 80 is deposited in the cavity 34 in any known manner, including manually. The adsorbent material 80 is preferably, but not necessarily, deposited in the cavity 34 in such a way that it substantially centers the plant embryo 42 within the cavity 34. Although it is preferred that the adsorbent material 80 substantially center the plant embryo 42 within the cavity 34, the plant embryo 42 need not be positioned that way. The adsorbent material 80 only needs to position the plant embryo 42 inside the cavity 34 in any known manner to place the plant embryo 42 in functional contact with the nutrient medium 26. In addition, it is not necessary for the adsorbent material 80 to "surround" the plant embryo 42. The adsorbent material 80 may In other embodiments within the scope of the appended claims, the adsorbent material 80 only needs to fill, either completely or partially, one or two sides of the space between the plant embryo 42 and the walls of the cavity. In one embodiment, the adsorbent material is 80 cavity 34 charcoal.
    Preferably, the charcoal is in the form of a powder and is activated by treatment with an acid, such as HCl or phosphoric acid. Activated charcoal is commercially available. Powdered activated charcoal of the NORIT® CNSP or DARCO® KB-G type is available from Norit Americas lnc., Marshall, Texas.
    In another embodiment, the adsorbent material 80 in the cavity 34 is a nutrient treated charcoal. An example of a process for producing nutrient-treated charcoal for insertion into the cavity 34 is described in Example 2.
    The term “plant embryo” as used herein refers to either a zygotic plant embryo or a somatic plant embryo. A zygotic plant embryo is an embryo that is found inside a botanical seed produced through sexual reproduction. Somatic embryos can be produced by culturing embryogenic tissue using standard methods under laboratory conditions in which the cells comprising the tissue are separated from each other and forced to develop into very small complete embryos.
    As used herein, the term "somatic plant embryo" refers to an embryo produced by culturing totipotent plant cells, such as meristem tissue, under laboratory conditions in which the cells comprising the tissue are separated from each other and forced to develop into small complete embryos.
    Alternatively, somatic embryos can be produced by induction of “cleavage polyembryogenesis” of zygotic embryos. Methods for producing somatic plant embryos suitable for use in the methods of the invention are standard in the art and have been previously described (see, e.g., U.S. Patent Nos. 4,957,866, 5,034,326, 5,036,007, 5,041,382, 5,236,841). , 5,294,549, 5,482,857, 5,563,061 and 5,821,126). For example, plant tissue can be grown in an initiation medium that includes hormones for initiating the formation of embryogenic cells, such as embryo suspension masses capable of developing into somatic embryos. The embryogenic cells can then be further cultured in a maintenance medium that promotes the establishment and proliferation of the embryogenic cells. Thereafter, the increased embryogenic cells can be cultured in a development medium that promotes the development of somatic embryos, which can also be subjected to post-developmental treatments, such as cold treatments. The somatic embryos used in the methods of the invention have completed the developmental stage of the somatic embryogenesis process. They may also have undergone one or more post-development treatments. plant embryos suitable for use in the methods of the invention may be derived from any plant species, such as di-hearted or mono-hearted plants, gymnosperms, etc. Conifer embryos suitable for use in the methods of the invention may be derived from coniferous types including, but not limited to, Embryos from yellow southern state stables ("Loblolly pine") and embryos from Douglas fir For use in produced seeds 20 of the present invention, the plant embryo 42 is usually sufficiently developed to have a shoot end and a root end.
    In some plant species, the shoot end includes one or more heart leaves at some stage of development. In other types of plants, the heart leaf or heart leaves are located elsewhere than at the shoot end. In another aspect, the present invention provides methods for improving the germination of a plant embryo based on a manufactured seed. in one embodiment, the method comprises the steps of (a) assembling a manufactured seed including a seed coat and a constraint, the constraint comprising a cavity; (b) adding nutrient medium including an adsorbent material to the seed coat, the adsorbent material being present in the medium at a concentration of from about 30 g / l to about 100 g / l; (c) placing a plant embryo in the cavity of the restriction; and (d) culturing the produced seed under conditions suitable for germination of the plant embryo.
    In one embodiment, the method comprises the steps of (a) assembling a manufactured seed including a seed coat and a constraint, the constraint comprising a cavity; (b) adding nutrient medium including an adsorbent material to the seed coat, wherein the adsorbent material is present in the medium at a concentration of from about 30 g / l to about 100 g / l; (c) placing a plant embryo in the cavity of the restriction; (d) adding an adsorbent material to the cavity; and (e) culturing the produced seed under conditions suitable for germination of the plant embryo.
    Conditions suitable for germination of manufactured seeds are standard in the field and include conditions suitable for germination of natural seeds. The produced seed can, for example, be sown in any of a number of environments, such as sand, vermiculite, sterile soil and / or in the field (natural soil). Sterile washed sand of the ColesTM type, which is available from a number of stores with garden products, can be used, for example.
    An example of a method of assembling a plant embryo 42 into a manufactured seed is described in Example 3.
    The methods of the invention improve the germination of a plant embryo starting from a manufactured seed, as shown in Examples 4, 5 and 6.
    The following examples are provided for the purpose of illustrating, but not limiting, the invention. 10 15 20 535 G10 14 EXAMPLES Example 1 This example shows a representative process for preparing a suitable nutrient medium for use in the invention.
    Complete nutrient medium (KE64-50) is prepared by combining the base medium KE64 (Table 1) with the components in Table 2, as described. KE64-50 is prepared based on pre-prepared storage solutions. The required amount of each storage solution (which is not heat labile) is added to water. Chemicals that do not originate from storage solutions (such as charcoal and agar) are weighed and added directly to the medium. After the addition of the non-heat-labile chemicals and compounds, the volume of the medium is adjusted to a suitable level, and the pH is adjusted to 5.7. The agent is then sterilized by autoclaving for 25 minutes.
    TABLE 12 FORMULATION OF THE BASIC MEDIA KE64 Medium grain Final concentration 301 1 10 0 0 06 299 1800 0 1000 0 6 0 - 0 ° 0 Ferric citrate 60 Pluronic F-68 10 18 Filter sterilized, heat-labile components (Table 2) are added after the medium has been cooled to 40 ° C. 10 535 CVIÜ 1 5 TABLE 2 Medium component Final concentration (mM) Final concentration (mg fl) _ Myoinositol 0.5549 100.0 Thiamine-HCl 0.0030 1.0 Pyridoxine-HCl 0.0012 0.25 Nicotinic acid 0.0081 1.0 Riboflavin 0.0021 0.125 Ca pantothenate 0.50 Biotin 0.0003 0.0010 Folic acid 0.8077 0.1250 L-asparagine 1, 8255 106.7 L-glutamine 0.3646 266.7 L-lysine-2HC | 0.7612 53.3 DL-serine 0.4631 80 L-proline 1.5310 53.3 L-arginine-HCl 0.4552 266.7 Urea 13.3200 800 L-valine 0.5983 53.3 L-alanine 0.2203 53.3 L-Ileucine 0.2448 80 L-threonine 0.3226 26.7 L-phenylalanine 0.1720 53.3 L-histidine 0.1308 26.7 L-tryptophan 02035 26.7 L-isoleucine 1.2930 26.7 L-methionine 0.7100 26.7 _L_-glycine 0.0003 53.3 L-tyrosine 0.2242 53.3 L-cysteine 0.6098 26.7 Sucrose 50 g / l Gibberillic acid (GAW ) 0.1 Antimicrobial substances 1.0 ml / ml Example 2 This example describes a representative process for the preparation of nutrient-treated charcoal for use in the invention. The base medium KE64 (Table 1) is prepared as described in Example 1, but without Pluronic F-68 and without agar. Nutrient-treated charcoal is prepared as follows: 23.3 g of charcoal of 100 mesh is added to 1 liter of base medium of type KE64. The components are autoclaved and allowed to cool to 40 ° C. The components of Table 2, as described in Example 1, are added sterile to the base medium KE64, and the medium is stirred to mix the components. The medium is filtered through Whatman No. 1 filter paper into a Buchner funnel to collect the charcoal. A moisture balance scale is used to determine the moisture content of the charcoal cake, and the dry weight of the charcoal is calculated. If the nutrient-supplied charcoal is to be added to the integrity of the manufactured seed, it is first dried until it becomes a surface material.
    Example 3 This example describes a representative process for assembling plant embryos into manufactured seeds and germination of manufactured seeds. In an example of a process for producing a manufactured seed for use in the invention, the seed coat is prepared by cutting a polycaprolactone hose to a suitable length. Ceramic shot barriers are made by injecting a porcelain strip into a preformed template with a needle in the center to provide the shot accepting cavity. The strip is allowed to dry to a consistency that allows removal of the preformed constraint.
    The confinement is then heated to a temperature which allows the porcelain to form a porous but fused structure. The restriction can be washed with acid to remove contaminants, if desired. The lids are made by pre-stretching ParafilmW '(Pechiney Plastic Packaging, Chicago, Illinois 60631).
    Zygotic embryos are prepared based on botanical seeds.
    The seeds are surface sterilized using methods similar to those described previously (Cyr et al, Seed Sci. Res. 1: 91-97 (1991)). The seeds are broken so that they open, and the zygotic embryos are dissected out of the megagamethophyte using a scalpel and tweezers in a fume hood with a laminar fl fate.
    Somatic embryos are prepared using standard methods previously described (see, e.g., U.S. Patent Nos. 4,957,866, 5,034,326, 5,036,007, 5,041,382, 5,236,841, 5,294,549, 5,482,857, 5,563). 061 and 5 821 126). For example, plant tissue can be grown in an initiation medium that includes hormones for initiating the formation of embryogenic cells, such as embryo suspensor masses capable of developing into somatic embryos. The embryogenic cells can then be further cultured in a residence medium that promotes the establishment and proliferation of the embryogenic cells. Thereafter, the proliferated embryogenic cells can be cultured in a development medium that promotes the development of somatic embryos, which can also be subjected to post-developmental treatments, such as cold treatments. The somatic embryos used in the procedures according to the invention have completed the development stage of the somatic embryogenesis process. They may also have undergone one or more of your post-development treatments.
    Manufactured seeds are assembled by thermobonding the ceramic shoot restriction 22 to the seed envelope 24. The seed envelope 24 is then filled with nutrient medium 26, and an embryo is inserted into the cavity 34 of the heart leaf restriction 22 with the heart leaf end first. Dry charcoal filler 80 (either nutrient treated or non nutrient treated) can be filled into the heart leaf restriction after the embryo has been inserted into the cavity 34. After the charcoal has been added, the seeds are then sealed using a second end closure by placing it over it. open the end of the seed and fuse the lids to the surface using heat. The primary end closures are dipped in a blue wax mixture before being attached to the other end closure. This promotes satisfactory bonding between the primary and secondary end connections. The seeds are then brushed with antimicrobial substances.
    An appropriate amount of sterile sand is prepared by burning 2 liters of sand at a temperature of 191 ° C (375 ° F) for 24 hours. The sand is then added to pre-sterilized trays, and 285 ml of water are added. Then grooves are created, and the box is closed. The box containing sand is then autoclaved for 1 hour at 121 ° C and a pressure of 1 atmosphere.
    The produced seeds are sown in the sand and allowed to germinate. The produced seeds are usually grown under continuous light at room temperature (23 ° C) for four to five weeks.
    Example 4 This example shows a representative method according to the invention for improving the germination of a plant embryo starting from a manufactured seed. Produced seeds were assembled as described in Example 3 but with the variations described below, and zygotic embryos of yellow southern state pine were introduced into the seeds (one embryo per seed). In each experiment below, charcoal was added to the base medium KE64, prepared as described in Example 1, prior to autoclaving. The concentration of charcoal in the medium was 2.5 g / l, 60 g / l or 100 g / l. In some treatments, the charcoal was nutrient treated, as described in Example 2. In some embodiments, the charcoal was not nutrient treated. After autoclaving, the rest of the components were added to prepare a complete KE64 medium.
    Two different shot restrictions, Type A and Type B, were used in the treatments. In some treatments, charcoal was also added to the cavity in the shot restriction. In some treatments, the charcoal added to the cavity was nutrient treated. In some treatments, the charcoal added to the cavity was not nutrient treated. In some treatments, charcoal was not added to the cavity. The seeds were then allowed to germinate as described in Example 3.
    The treatments are described below.
    Fabric A of shot restriction Treatment 1: Complete KE64 medium + 2.5 g / l non-nutrient-treated charcoal Treatment 2: Complete KE64 medium + 60 g / l non-nutrient-treated charcoal Treatment 3: Complete KE64 medium + 60 g / l nutrient-treated charcoal Treatment 4: Complete KE64 medium + 2.5 g / l non-nutrient-treated charcoal, and nutrient-treated charcoal in the cavity Treatment 5: Complete KE64-medium + 60 g / l non-nutrient-treated charcoal, and nutrient-treated charcoal in the cavity Treatment 6: Complete KE64 medium + 60 g / l nutrient-treated charcoal, and nutrient-treated charcoal in the cavity Treatment 7: Complete KE64-medium + 2.5 g / l non-nutrient-treated charcoal, and non-nutrient-treated charcoal in the cavity Fabric B of shot restriction Treatment 8: Complete KE64 medium + 2.5 g / l non-nutrient-treated charcoal, and nutrient-treated charcoal in 10 15 20 25 535 CV10 19 cavity Complete KE64 medium + 60 g / l non-nutrient-treated charcoal, oc h nutrient-treated charcoal in the cavity Treatment 9: Treatment 10: charcoal, and nutrient-treated charcoal in the cavity RESULTS Data were collected 28 days after sowing. Several parameters were measured to determine the effects of increasing the amount of charcoal in the nutrient medium and / or adding charcoal to the cavity of the shot restriction. The lengths of the rootstock, hypocotylene, heart leaves and epicotylene were measured. The term "rootstock" refers to the portion of the plant embryo that develops into the primary root of the resulting plant. The term "heart leaf" generally refers to the first, first pair of or first wreath (depending on the type of plant) of leaf-like structures on the plant embryo which primarily serve to make nutrient compounds in the seed available to the developing embryo, but function in some cases as nutrient stores or photosynthesis structures. The term "hypocotyl" refers to the part of a plant embryo or seedling that is located below the heart leaves, but above the rootstock. The term "epicotyl" refers to the part of the seedling stem that lies above the heart leaves.
    The organ lengths were measured in centimeters and are shown in Table 3.
    The degree of germination was measured and shown in Table 4. The normality of the germs was also assessed and shown in Table 5. The embryos were examined and classified as normal; would be normal if they were completely extracted from the cavity; abnormal; completely extracted from the cavity, but not normal; and unchanged. The term "normal germ" or "normality" denotes the presence of all expected parts of a plant at the time of evaluation. In the case of gymnosperms, the normality is characterized by the root substance having a length of more than 3 mm and no visible discernible malformations compared to the appearance of embryos that are brought to germinate on the basis of a natural seed. "Abnormal" means that the tissue of at least one organ is swollen, and that the root and heart leaves are dead. "Completely extracted, but abnormal" means that the germ has come out completely and poured Complete KE64 medium + 60 g / l nutrient-treated 535 010 20 from the high, but is abnormal. "Unchanged" means that the embryo has not changed from day one for the experiment.
    Table 3: Organs (measured in cm) Treatment Root substance d <0.0001 "Hypocotyl d = 0, 2788 Hjänbiad u = 0, 1 174 Epikoiyi a = 0.2o74 Type A of restriction Medium: 2.5 g / l ej nutrient-treated charcoal Holicity: no charcoal 1,035 2.02 1,16 0,43 Medium: 60 g / l non-nutrient-treated charcoal Holicity: no charcoal 1,35 “3,81 1,26 0,43 Medium: 60 g / l nutrient-treated charcoal Holiness: No charcoal 1,465 ”2,21 1,3 0,43 Medium: 2.5 g / l non-nutrient-treated charcoal Holiness: Nutrient-treated charcoal 1,14” 1,91 1, 21 Medium: 60 g / l non-nutrient-treated charcoal Cohesion: Nutrient-treated charcoal 1 34 “'° <° 2.46 1.32 0.13 Medium: 60 g / l nutrient-treated charcoal Holicity: Nutrient-treated charcoal 1 '54A, B, C, D 2.71 1.41 0.52 Medium: 2.5 g / l non-nutrient-treated charcoal Cohesion: Non-nutrient-treated charcoal 1 94% ”2.63 1.77 0, 28 Type B of restriction Medium: 2.5 g / l non-nutrient-treated charcoal Holicity: Nutrient-treated charcoal 1 s9 ^ '“- ° 2.52 1.42 0.42 Medium: 60 g / l non-nutrient-treated charcoal Holiness: Nutrient-treated charcoal 2.20 ”2.88 1.47 0.16 10 Medium: 60 g / l nutrient-treated charcoal Holicity: Nutrient nes- treated charcoal 2.32 ^ 2.62 1.55 0.16 5 Followed by the same letter: no significant deviation 535 (HO 21 Table 4: Gradient degree Treatment Over1 Partiellz Unders Upside-down * d = 0.1310 G = 0 , Ö10O (l = 0.0022 = 0.3457 Type A of restriction 1 Medium: 2.5 g / l non-nutrient 13.9% 19.4% 55.6% 8.3% substance-treated charcoal Holicity: no charcoal 2 Medium: 60 g / l non-nutritional 41.7% 22.2% 27.8% 5.6% substance treated charcoal Cohesion: no charcoal 3 Medium: 60 g / l nutritional 36.1% 11.1% 44.4% 2.8% substance-treated charcoal Cohesion: No charcoal 4 Medium: 2.5 g / l non-nutrient 11.1% 8.3% 77.8% 0% substance-treated charcoal Habitat: Nutrient-treated charcoal 5 Medium: 60 g / l non-nutrient 22.2% 16.7% 55.6% 5.6% substance-treated charcoal Holicity: Nutrient-treated charcoal 6 Medium: 60 g / l nutrient- 19.4% 16.7% 61, 1% 0% substance treatment lat charcoal Coverage: Nutrient-treated charcoal 7 Medium: 2.5 g / l non-nutrient 30.6% 33.3% 30.6% 2.8% substance-treated charcoal Holicity: Non-nutrient-treated charcoal Type B of restriction 8 Medium: 2.5 g / l non-nutrient 38.9% 27.8% 30.6% 0% substance-treated charcoal Holicity: Nutrient-treated charcoal 9 Medium: 60 g / l non-nutrient 36.1% 25% 36 , 1% 2,8 ° /> carbon-treated charcoal Cohesion: Nutrient-treated charcoal 10 Medium: 60 g / l nutrient 33.3% 25% 36.1% 2,8% carbon-treated charcoal Cohesion: Nutrient-treated charcoal "Over "refers to heart leaves and hypocotyl above ground" "Partial" refers to certain green tissue that appears above the ground "Below" means that nothing appears above the ground "Upside down" means that the root has died and that the hypocotyl has been turned up in the air 535 C110 22 Table 5: Normality Treatment Normal Would be Abnormal Fully- Abnormal abnormally extra- changed if they were hered, completely abnormal extracted- a = 0.0206 u = 0.1325 a = 0.6576 d = 0.4540 a = 0.1064 Type A: Limitation 1 Medium: 2.5 g / l not 13.9% 5.6% 27.8% 5.6% 44.4% nutrient-treated charcoal Cohesion: Longcoat 2 Medium: 60 g / l not 36.1% 19.4% 25% 0% 19.4% nutrient-treated charcoal Ha @ et: Own charcoal 3 Medium: 60 g / l 36.1% 5.6% 25% 0% 27.8% nutrient-treated charcoal Highness : l fl çt charcoal 4 Medium: 2.5 g / l not 8.3% 8.3% 25% 0% 58.3% nutrient-treated charcoal Holicity: Nutrient-treated charcoal 5 Medium: 60 g / l not 16.7 % 8.3% 36.1% 0% 36.1% nutrient-treated charcoal Cavity: Nutrient-treated charcoal 6 Medium: 60 g / l 16.7% 13.9% 16.7% 0% 50% nutrient-treated treated charcoal Holiness: Nutrient-treated charcoal 7 Medium: 2.5 g / l not 33.3% 22.2% 19.4% 2.8% 22.2% nutrient-treated charcoal Holicity: Non-nutrient-treated charcoal Type B: Limitation 8 Medium: 2.5 g / l not 36.1% 16.7% 13.9% 0% 27.8% nutrient-treated charcoal Cavity: Nutrient-treated charcoal 10 15 20 535 Ü'IÜ 23 Treatment Normal Would be Abnormal Full- Unfor- normal di gt extra- changed if they were hered. completely abnormal extracted G = 0.0206 a = 0.1325 cr = 0.6576 d = 0.4540 a = 0.1064 9 Medium: 60 g / l not 41.7% 8.3% 13.9% 2.8% 33.3% nutrient-treated charcoal Cavity: Nutrient-treated charcoal 10 Medium: 60 g / l 27.8% 30.6% 16.7% 0% 25% nutrient-treated charcoal Cavity: Nutrient-treated charcoal The values in Tables 3, 4 and 5 generally show an increase in organ length, germination rate and normality of embryos as a cave based on produced seeds, in which the nutrient includes a high content of charcoal (60 g / l compared to 2.5 g / l l).
    Example 5 This example shows a representative method according to the invention for improving the germination of a plant embryo based on a manufactured seed. Produced seeds were assembled as described in Example 3, but according to the variations described below, and zygotic embryos of yellow southern state pine were introduced into the seeds (one embryo per seed). In each treatment below, charcoal was added to base media KE64, prepared as described in Example 1, prior to autoclaving. The concentration of charcoal in the medium was 0 g / l, 60 g / l or 100 g / l.
    In some treatments, the charcoal was nutrient treated and prepared as described in Example 2. In some treatments, the charcoal was not nutrient treated. After autoclaving, the rest of the components were added to prepare complete KE64 medium.
    Only Type B shot restrictions were used in the treatments. In all treatments, nutrient-treated charcoal was added to the cavity in the shot restriction. The seeds were then allowed to germinate, as described in Example 3. The treatments are described below.
    Treatment 1: Complete KE64 medium; and nutrient-treated Treatment 2: Treatment 3: Treatment 4: Treatment 5: RESULTS 535 010 24 charcoal in the whole Complete KE64 medium + 60 g / l non-nutrient-treated charcoal; and nutrient-treated charcoal in the cavity Complete KE64 medium + 60 g / l nutrient-treated charcoal; and nutrient-treated charcoal in the whole Complete KE64 medium + 100 g / l non-nutrient-treated charcoal; and nutrient-treated charcoal in the cavity Complete KE64 medium + 100 g / l nutrient-treated charcoal; and nutrient-treated charcoal in the holiness Data were collected 41 days after sowing. Several parameters were measured to determine the effects of increasing the amount of charcoal in the nutrient medium and adding charcoal to the cavity of the shot restriction. Organ lengths were measured, and the degree of germination and normality were determined. These terms are defined as in Example 4. The organ lengths are shown in Table 6, the degree of germination is shown in Table 7 and the normality is shown in Table 8.
    Table 6: Organ lengths (measured in cm) Treatment Root substance d = 0.0200 Hypocotyl d = 0.0192 Heart rate a = 0.11s3 Epicotyl <1 = 0.041 5 1 Medium: no charcoal Holicity: Nutrient-treated charcoal 1.95 ”2 , 41 ”1.44 0.375 Medium: 60 g / l non-nutrient-treated charcoal Holicity: Nutrient-treated charcoal 2.57” 2, 62 ^ B 1.55 0.43 ”Medium: 60 g / l nutrient-treated charcoal Holiness: Nutrient-treated charcoal 2.74 ”2.9s ^ 1.69 0.41” Medium: 100 g / l non-nutrient-treated charcoal Holiness: Nutrient-treated charcoal 2.56 ”2.78” 1.75 0.62 ^ Medium: 100 g / l nutrient-treated charcoal Cohesion: Nutrient-treated charcoal 2.99 ^ 2.70 ”1.61 0, 50 ^ B 535 Ü'l0 25 Table 7: Degree of growth Beham ham mg q-Égïß aïärltlïz a-Uonïïgs Uåff-Joåï-: ftsar 1 Medlumnngeufäkol 57.4% “224% 10% 1o, 2% ^ Hålighet: Nutrient-treated charcoal 2 Mediurfueog / lejnäfangs- 571%” 314% 10% 1.4% “bill-treated charcoal Cavity: Nutrient-treated charcoal a Medium; so g / l nutrient- 77, s% ^ 15.7% ^ 5.2% 14% ”substance-treated charcoal Cavity: Nutrient-treated charcoal 4 Medium: 1009/15; 5a, 5 ° /.,^ “343% 45% 2,9% B nutrient-treated charcoal Cavity: Nutrient-treated charcoal 5 Meaiumnoo g / lnärings- 72,5%“ 2o.3% 4,31% 2,9% “ substance-treated charcoal Cavity: Nutrient-treated charcoal Table 8: Normal Normal Would Abnormal Unchanged Vala HOT- template if it were completely extracted! Treat u = 0.0345 u = 0.0192 c = 0., 1183 u = 0.0415 1 Mediumnngeufäkol 559% ”s, a% 2o, a% 5.57., Cavity: Nutrient-treated charcoal 2 Medium: 50 g /smile; nutrient 72,970 ”56% 129% 5.7% substance-treated charcoal Cavity: Nutrient-treated charcoal 3 Medium: eo g / l nutrient-ao, 5%” 1o, o% ^ 57% 29% substance-treated charcoal Cavity: nutrient-treated charcoal 4 Medium: 1oo g / lejnärings- 72.9 ° /, ^ “17.1% 5,15% o, o% substance-treated charcoal Cavity: Nutrient-treated charcoal s Mediumnoog / lnäfings- s4, s% ^ 7,1 % 43% 4.3% substance-treated charcoal Cavity: Nutrient-treated charcoal 10 15 20 25 535 Ü1Ü 26 The values in Tables 6, 7 and 8 show an increase in organ length, germination rate and normality in embryos that have germinated on the basis of manufactured seeds, in which the nutrient medium includes a high content of charcoal (60 g / l compared to 0 g / l).
    The values in Table 7 also show a reduced degree of germs of the "up-and-down" type when the amount of charcoal in the medium increases.
    Example 6 This example shows a representative method according to the invention for improving the germination of a plant embryo based on a manufactured seed. Produced seeds were assembled as described in Example 3, but subjected to the variations described below, and zygotic embryos of yellow southern state stables were introduced into the seeds (one embryo per seed). In each experiment below, charcoal was added to the base medium KE64, prepared as described in Example 1, prior to autoclaving. The volume of the base medium was adjusted before adding the charcoal. The concentration of charcoal in the medium was 0 g / l, 3 g / l, 30 g / l, 50 g / l, 60 g / l, 75 g / l or 100 g / l. In all experiments, the charcoal was nutrient treated and prepared as described in Example 2, except that no sucrose was added to the base medium KE64 in the preparation of the nutrient treated charcoal for this experiment. After autoclaving, the rest of the components were added to prepare complete KE64 medium (II), which differs from ordinary complete KE64 medium in that the sucrose concentration is 63.5 g / l.
    Only Type A shot restrictions were used in the experiments. In all treatments, nutrient-treated charcoal was added to the cavity in the shot restriction. The seeds were then allowed to germinate as described in Example 3.
    The treatments are described below. Each treatment was performed with an average of 60 seeds per treatment.
    Treatment 1: Complete KE64 medium (ll) + 100 g / l nutrient-treated charcoal Treatment 2: Complete KE64 medium (ll) + 75 g / l nutrient-treated charcoal Treatment 3: Complete KE64 medium (ll) + 60 g / l nutrient-treated charcoal Treatment 4: Complete KE64 medium (ll) + 50 g / l nutrient-treated charcoal 10 15 20 535 Ü'i0 27 Treatment 5: Complete KE64 medium (ll) + 30 g / l nutrient - treated charcoal Treatment 6: Complete KE64 medium (ll) + 3 g / l nutrient-treated charcoal Treatment 7: Complete KE64-medium (II) + 0 g / l nutrient-treated charcoal RESULTS Data were collected on days 19, 26 and 35 after sowing. Several parameters were measured to determine the effects of increasing the amount of charcoal in the nutrient medium and adding charcoal to the cavity of the shot restriction.
    Organ lengths were measured, and the degree of germination and normality were determined. The terms are defined as in Example 4. The degree of germination is shown in FIGURES 2 and 3, the organ lengths are shown in FIGURE 4 and the presence of normality is shown in FIGURE 5.
    On day 19, embryos in seeds with a higher charcoal content in the nutrient medium showed faster germination than seeds with a lower charcoal content (FIGURE 2). On day 26, the seeds with a higher charcoal content maintained a higher degree of germination (FIGURE 3).
    In addition, organ lengths were generally greater in embryos in seeds with more charcoal in the nutrient medium (FIGURE 4). Finally, nonnallet data showed a higher prevalence of normal germs from embryos in seeds with more charcoal in the nutrient medium (FIGURE 5). On the other hand, the degree of inactive embryos and germs that would be normal if they were extracted completely increased with decreasing charcoal content. These data suggest that increasing the amount of charcoal in the nutrient medium improves the performance of embryos in the seed produced. Although the preferred embodiment of the invention has been elucidated and described, it will be appreciated that various changes may be made therefrom without departing from the spirit and scope of the invention.
    权利要求:
    Claims (18)
    [1]
    Manufactured seed comprising: (a) a seed coat, (b) a nutrient medium comprising an adsorbent material, the adsorbent material being charcoal, polyvinylpolypyrrolidone or silica gels and present in the medium in a concentration of from about 30 g / l to about 100 g / l; and (c) a shot restriction, the shot restriction including a cavity.
    [2]
    Manufactured seed according to claim 1, wherein the adsorbent material is charcoal.
    [3]
    Manufactured seed according to claim 2, wherein the charcoal is present in the medium in a concentration of from about 50 g / l to about 100 g / l.
    [4]
    Manufactured seed according to claim 2, wherein the charcoal is nutrient treated.
    [5]
    Manufactured seed according to claim 2, wherein the charcoal is not nutrient treated.
    [6]
    A seed produced according to claim 1, further comprising a plant embryo located in the cavity of the shoot restriction.
    [7]
    Manufactured seed according to claim 6, further comprising an adsorbent material in the cavity of the shoot restriction.
    [8]
    Manufactured seed according to claim 7, wherein the adsorbent material in the cavity is charcoal.
    [9]
    Manufactured seed according to claim 8, wherein the charcoal is nutrient treated. 10 15 20 25 30 535 Û'l0 29
    [10]
    Manufactured seed according to claim 8, wherein the charcoal is not nutrient-treated.
    [11]
    A method of germinating a plant embryo starting from a manufactured seed, comprising: (a) assembling a manufactured seed comprising a seed coat and a shoot restriction, the shoot restriction comprising a cavity; (b) adding nutrient medium including an adsorbent material to the seed coat, the adsorbent material being charcoal, polyvinylpolypyrrolone or silica gels and present in the medium at a concentration of from about 30 g / l to about 100 g / l; (c) placing a plant embryo in the cavity of the shoot restriction; and (d) culturing the produced seed under conditions suitable for germination of the plant embryo.
    [12]
    The method of claim 11, wherein the adsorbent material is charcoal.
    [13]
    A method according to claim 12, wherein the charcoal is nutrient treated.
    [14]
    A method according to claim 12, wherein the charcoal is not nutrient treated.
    [15]
    The method of claim 11, further comprising adding an adsorbent material to the cavity of the shot restriction.
    [16]
    The method of claim 15, wherein the adsorbent material added to the cavity is charcoal, polyvinylpolypyrroledone or silica gels.
    [17]
    The method of claim 16, wherein the adsorbent material added to the cavity is charcoal.
    [18]
    A method according to claim 17, wherein the charcoal added to the cavity is nutrient treated.
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    同族专利:
    公开号 | 公开日
    AU2010219404A1|2011-04-14|
    CN102027821A|2011-04-27|
    US20110072716A1|2011-03-31|
    US8375630B2|2013-02-19|
    SE1051008A1|2011-03-31|
    BRPI1003915A2|2013-01-29|
    CA2713626C|2013-11-19|
    FI20106003A|2011-03-31|
    CA2713626A1|2011-03-30|
    AR078409A1|2011-11-02|
    NZ587898A|2012-03-30|
    UY32909A|2011-04-29|
    FI125760B|2016-02-15|
    FI20106003A0|2010-09-28|
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    法律状态:
    2018-05-02| NUG| Patent has lapsed|
    优先权:
    申请号 | 申请日 | 专利标题
    US24735409P| true| 2009-09-30|2009-09-30|
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