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    • 专利摘要:
    SUMMARY A process for producing an improved nutrient medium comprising an adsorbent material for growing plant embryos is described.
    公开号:SE535083C2
    申请号:SE1051015
    申请日:2010-09-30
    公开日:2012-04-10
    发明作者:Jeffrey E Hartle；William C Carlson
    申请人:Weyerhaeuser Nr Co；
    IPC主号:
    专利说明:

    The composition of one or more components of a first nutrient medium after incubation with a desired amount of adsorbent material; and (b) preparing an improved nutrient medium comprising the same components as the first nutrient medium, the improved nutrient medium comprising: (i) an increased concentration of one or more components determined in step (a) to have a reduced concentration in the presence of the absorbent material; and (ii) the same type of absorbent material in a concentration range that is twofold relative to that used in step (a).
    According to another aspect of the invention there is provided a process for the preparation of an improved nutrient medium comprising an adsorbent material for culturing plant cells. The method of this aspect of the invention comprises (a) incubating a first nutrient medium comprising a predetermined initial concentration of components comprising one or more carbon sources, vitamins, minerals and amino acids with a desired amount of adsorbent material for addition to an improved nutrient medium; (b) determining whether there is a reduction in the concentration of one or more of the components of the first nutrient medium after the incubation in step (a) compared to the predetermined initial concentration of the component; and (c) preparing an improved nutrient medium comprising the same components as the first nutrient medium, the improved nutrient medium comprising: (i) an increased concentration of said one or more components which in step (b) was determined to have a reduced concentration in the presence of the adsorbent material; and (ii) the same type of adsorbent material in a concentration range that is at most twice as high as that used in step (a).
    The methods for producing an improved nutrient medium are useful for the growth and / or germination of a plant embryo, such as a conifer embryo.
    DESCRIPTION OF THE DRAWING The above aspects and many of the attendant advantages of the present invention will be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings. FIGURE 1 shows in cross-sectional view an example of a manufactured seed comprising a plant embryo according to various embodiments of the invention.
    DETAILED DESCRIPTION Using the present description section, manufactured seeds including a modified nutrient medium are provided, resulting in an improved germination rate compared to manufactured seeds including a standard nutrient medium containing nutrient-treated charcoal.
    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 very small complete embryos. Methods for the production of 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). 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 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 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.
    Usually the plant embryos used according to the invention have a shoot end and a root end. In some plant species, the shoot end includes one or more heart leaves (leaf-like structures) at a certain stage of development. Plant embryos suitable for use in the methods of the invention may be derived from any plant species, such as di-cardiac or mono-cardiac plants, gymnosperms, such as zygotic or somatic embryos (eg pine, such as yellow For use in manufactured seeds 20 of the present invention, the plant embryo usually develops sufficiently to have a shoot end and a root end. other types of plants are the heart leaf or heart leaves located elsewhere than at the shoot end.
    The term “germination” as used herein refers to a physiological process which results in the elongation of a plant embryo along its axis, and this is complete when the embryo has been extended to the point where it protrudes from the seed coat or the lid of the manufactured seed.
    The term “complete germination” as used herein refers to a manufactured seed whose root has penetrated through the seed coat or the lid of the manufactured seed.
    A seed produced for use in the invention includes a plant embryo, a casing for the seed produced and a nutrient medium. 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 comprising a plant embryo, a casing of a manufactured seed enclosing the somatic plant embryo and comprising an orifice, a nutrient medium in functional contact with the plant embryo and a lid closing the somatic plant embryo within the produced seed; however, the method 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 a seed coat 24, a nutrient medium 26, a bottom closure 28 and an optional cylcap ("cylcap") 22 (shoot limitation). In the exemplary embodiment shown in FIGURE 1, the produced seed 20 includes a plant embryo 42, a seed coat 24, a nutrient medium 26 in functional contact with the plant embryo, and an optional cylcap 22 (shoot restriction).
    As used herein, the term "manufactured seed coat" refers to a structure analogous to that of a natural seed coat which protects the plant embryo and other internal structures of the manufactured seed from mechanical damage, dehydration, attack of microbes. , fungi, insects, nematodes, birds and other pathogens, herbivores and pests, to name a few. polymer 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. and resistant to the penetration of plant pathogens until after the emergence of the germinating embryo. terial. The seed coat can be a cut straw made of fibrous material, such as paper. The suction pipe parts can be pre-treated in a suitable coating material, such as wax. Alternatively, the seed coat can be formed on the basis of a tubular part of a biodegradable plastic material. One such material is polylactic acid ("PLA"), sold by MAT-UR in Los Angeles, California. Oklahoma 43612) with or without a plasticizer in the form of 1% Tegomer HS | 6440 (Degussa Goldschmldt Chemical Corp., 914 East Randolph Road, Hopewell, Virginia 23860). Such biodegradable plastic pipes require or do not require a wax coating, since such pipes are already resistant to surrounding elements. Additives, such as antibiotics and plant growth regulators, may, for example, be added to the seed coat 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. .
    Said cylcap 22, also known as shoot restriction or heart leaf restriction, 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 heart leaf 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 gametophyte medium). The heart blade restraining portion 32 is suitably integrally or uniformly shaped together with the end closure portion 30. The cylcap 22 also includes a longitudinally extending cavity 34 extending through the end closure portion 30 and partially through one end of the heart blade restraining portion 32. The open end 34 is known as the cavity. as a heart leaf restriction opening 36. The cavity 34 is sized to receive a plant embryo 42 therein. As shown in FIGURE 1, said cylcap 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 provide a conditioned embryo, as described herein.
    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, cementitious 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.
    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 the nutrient medium 26, providing a plurality of pathways. for the nutrients from the nutrient medium 26 to reach the embryo 42. 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 in this manner.
    Adsorbent material 80 need only position the embryo 42 within the cavity 34 in any known manner to put the plant embryo 42 in functional contact with the nutrient medium 26. In addition, in certain embodiments of the invention, the filler material 80 need only fill a or two sides, either completely or partially, of the space between the embryo 42 and the walls of the cavity 34.
    Preferably, the filler material 80 is an adsorbent, such as activated charcoal, Dowex resins, zeolites, alumina, clay, diatomite, silica gel and kieselguhr.
    During assembly of the manufactured seed 20, the filler material 80 is placed in the cavity 34 of the cylcap 22 in any manner known in the art, including manually. The filling material 80 is preferably, but not necessarily, placed in the cavity 34 in such a way that it substantially centers embryos 42 in the cavity 34. The centering of the embryo 42 in the cavity 34 increases the surface area of the embryo 42 which is in functional contact with the nutrient medium 26.
    The term "functional contact" as used herein is intended to mean a position in which the embryo 42 absorbs nutrients from the nutrient medium 26.
    In some embodiments, the filler material is 80 charcoal. Preferably, the charcoal is in the form of a powder and is activated by pretreatment with an acid, such as HCl or phosphoric acid. Activated charcoal is commercially available.
    Powdered activated charcoal of the type NORIT® CNSP or DARCO® KB-G is produced by chemical activation using a phosphoric acid process and is available from Norit Americas Inc., Marshall, Texas, 75671.
    In some embodiments, the filler material 80 is nutrient treated charcoal. The term "nutrient-treated" charcoal as used herein refers to charcoal which has been in contact with a medium containing a variety of nutrients, such as a carbon source, vitamins, minerals and amino acids, in such a way that the charcoal absorbs and conserves the nutrients from the medium. The representative medium used to produce nutrient-treated charcoal is the medium KE64, as described in Example 1. An example of a process for producing nutrient-treated charcoal for use as a filler 80 for insertion into the cavity 34 is provided in Example 1.
    In the seeds produced and in the processes according to the invention, the nutrient medium 26 (otherwise referred to as "gametophyte medium") is in functional contact with the plant embryo placed in the manufactured seed 20. The term "nutrient medium" used herein refers to a source of nutrients, such as vitamins. , minerals, carbon and energy sources, and other beneficial compounds used by the embryo during germination, thus the nutrient medium is analogous to the gametophyte of a natural seed.
    According to one aspect of the invention there is provided a process for the preparation of an improved nutrient medium comprising an adsorbent material for culturing plant cells. The method of this aspect of the invention includes (a) determining whether there is a reduction in the concentration of one or more components in a first nutrient medium after incubation with a desired amount of adsorbent material; (b) preparing an improved nutrient medium comprising the same components as the first nutrient medium, the improved nutrient medium comprising: (i) an increased concentration of said one or more components which in step (a) was determined to have a reduced concentration in the presence of the adsorbent material; and (ii) the same type of adsorbent material in a concentration range that is at most twice as high as that used in step (a).
    According to another aspect of the invention there is provided a process for the preparation of an improved nutrient medium comprising an adsorbent material for culturing plant cells. The method of this aspect of the invention comprises (a) incubating a first nutrient medium comprising a predetermined initial concentration of components comprising one or more carbon sources, vitamins, minerals and amino acids, with a desired amount of adsorbent material for addition to an improved nutrient medium; (b) determining whether there is a reduction in the concentration of one or more of the components of the first nutrient medium after the incubation according to step (a) compared to the predetermined initial concentration of the component; and (c) preparing an improved nutrient medium comprising the same components as the first nutrient medium, the improved nutrient medium comprising: (i) an increased concentration of said one or more components determined in step (b) to have a reduced concentration in the presence of the adsorbent material; and (ii) the same type of adsorbent material in a concentration range that is at most twice as high as that used in step (a).
    The methods of the invention are useful for preparing an improved nutrient medium including an absorbent material, such as charcoal, for use in the growth and / or germination of plant embryos. The improved nutrient medium prepared according to the invention is useful for the production and germination of manufactured seeds in a number of different contexts.
    According to the various aspects of the invention, a first nutrient medium including a predetermined initial concentration of components including one or more carbon sources, vitamins, minerals and amino acids is incubated with an absorbent material for a period of time sufficient for the various components of the medium to be adsorbed to the adsorbent material. . Suitable time periods for incubating the first nutrient medium with the adsorbent composition range from at least about 10 minutes up to fl days or a week or more, such as at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 8 hours, up to 24 hours, 48 hours or longer.
    Suitable adsorbent materials for use in the processes for preparing a modified (or improved) nutrient medium include, but are not limited to, charcoal, polyvinylpolypyrrolidone, and silica gels. In some embodiments, the adsorbent material in the first and modified (improved) nutrient medium is from 1.0 g / l to 100 g / l charcoal. In some embodiments, the charcoal added to the first and modified (improved) nutrient medium from 1.0 g / l to 100 g / l is non-nutrient treated charcoal (eg from 5 g / l, 20 g / l to 100 g / l, from 50 g / l to 100 g / l, from 60 g / l to 100 g / l, or from 50 g / l to 80 g / l, or about 60 g / l). The term "non-nutrient treated" charcoal as used herein refers to charcoal (eg pure charcoal or activated charcoal) which has not been contacted with a medium containing a plurality of nutrients, such as a carbon source, vitamins, minerals and amino acids, wherein Charcoal absorbs and preserves nutrients from the medium.
    According to this aspect of the invention, the first and modified (improved) nutrient medium includes the same type of adsorbent composition (eg charcoal). The concentration of adsorbent composition in the improved nutrient medium is usually within a concentration range that is about two to five times the ratio of the adsorbent incubated in the first nutrient medium. In certain embodiments of the process, the concentration of the adsorbent composition in the improved nutrient medium is the same as the concentration of adsorbent incubated in the first nutrient medium.
    After the first nutrient medium has been incubated with the adsorbent composition of step (a), an assay is performed to determine whether there is a reduction in the concentration of one or more of the components in the first nutrient medium after incubation compared to the initial concentration. of the component of the first nutrient medium. TABLES 3 and 4 show, for example, a comparison of the medium components before and after incubation with charcoal. For the one or fl components of the nutrient medium that have been determined to be reduced with respect to the concentration in the first nutrient medium after incubation with the adsorbent material, an adjustment is made to increase the concentration of the one or fl components of the improved nutrient medium. In certain embodiments of the process, an adjustment is made so that the increase in concentration of the component in the improved nutrient medium corresponds to the reduction in concentration observed in the first nutrient medium after incubation with the adsorbent composition. In certain embodiments of the process, the adjustment is made to the concentration of the component in the improved nutrient medium which also takes into account at least one of (1) the effect of the increased concentration of the particular component on the total pH of the medium; (2) the interaction with other components of the medium (ie precipitation); or (3) a maximum content of a particular component with respect to the viability of the plant embryo to be brought into contact with the nutrient medium.
    In certain embodiments, the process according to this aspect of the invention is carried out in preparation for scaling up in such a way that the first nutrient medium incubated with the adsorbent composition of step (a) has a volume of about 1/4 to 1/100 (such as 1 / 5, 1/10, 1/50, 1/75 up to t / 100) of the total volume of the improved nutrient medium according to step (c).
    In certain embodiments, the method further comprises placing the first nutrient medium from step (a) in a first set of manufactured seeds and placing the improved nutrient medium from step (c) in a second set of manufactured seeds, placing a conifer embryo in functional contact with the nutrient medium in each of the produced seeds from the first and second sets of produced seeds, placement of the produced seeds in an environment leading to plant growth and comparison of the germination frequencies of the embryos from the first and second sets of manufactured seeds to determine if there is an effect of the improved nutrient medium on the germination rate.
    According to this aspect of the invention, the first nutrient medium and the modified (improved) nutrient medium usually include the same components, the modified (improved) nutrient medium including an increased concentration of at least one or more of the components compared to the first nutrient medium. In certain embodiments of the process, the first and modified nutrient media include at least two components selected from the group consisting of NH 4 NO 3, KH 2 PO 4, myoinositol, thiamine-HCl, pyridoxine-HCl, nicotinic acid, ribovin, calcium pantothenate, biotin and folic acid. , DL-serine, L-proline, L-arginine-HCl and L-alanine.
    The nutrient medium usually also includes CuCl 2, CaCl 2, MgSO 4, ferric citrate, MnCl 3, H 3 BO 3, ZnSO 4 and (NH 4) 2 MoO 4, as described with reference to the medium designated "MSO 9", as described in Examples 1, 3 and 4.
    In some embodiments, the improved nutrient medium includes FeSO 4 at a concentration of from about 5 mg / L to 25 mg / L, such as from about 10 mg / L to about 15 mg / L. In some embodiments, the improved nutrient medium includes MgSO 4 at a concentration of from about 600 mg / L to about 1500 mg / L, such as from about 800 mg / L to about 1200 mg / L.
    The nutrient medium may additionally include amino acids. Suitable amino acids may include amino acids which are usually incorporated into proteins, as well as amino acids which are not usually incorporated into proteins, such as arginine succinate, citrulline, canavanine, ornithine and D-stereoisomers. In one embodiment, the nutrient medium also includes at least one amino acid selected from the group consisting of from 85 mg / L to 100 mg / L DL-serine; from 55 mg / l to 70 mg / l L-proline, from 300 mg / l to 600 mg / l L-arginine-HCl and from 55 mg / l to 70 mg / l L-alanine.
    The nutrient medium usually also includes one or more of your carbon sources, vitamins and minerals. Suitable carbon sources include, but are not limited to, monosaccharides, disaccharides and / or starches. The modified nutrient medium may also include one or more components involved in nitrogen metabolism, such as urea or polyamines.
    The nutrient medium may include oxygen carriers to enhance both the absorption of oxygen and the retention of oxygen in the nutrient medium, which enables the medium to maintain an oxygen concentration higher than that which would otherwise be present in the medium solely due to the absorption of oxygen from the atmosphere. Examples of oxygen carriers include perfluorocarbons, such as FC-77, and surfactants, such as Pluronic F-68, available from BASF Corp., Parsippany, N.J. Examples of oxygen carriers are described in U.S. Patent No. 5,564,224 (e.g., column 9, line 44, to column 11, line 67), which is hereby incorporated by reference.
    The nutrient medium 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.
    Biol. 49: 199-123 (1998)). Auxins are plant growth hormones that promote cell division and growth. Examples of auxins for use in the growth medium include, but are not limited to, 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, indole-3-butyric acid, naphthalene acetic acid and chlorogenic 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, β-furfurylaminopurine, dihydrogenzeatin, zeatin, kinetin and zeatin riboside. Gibberellins are a class of diterpenoid plant 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 4/7). , such as the type of 4/7 gibberellin sold by Abbott Laboratories, Chicago, Illinois. When abscisic acid is present in the modified nutrient medium, it is usually used in a concentration in the range of from about 1 mg / l to about 200 mg / l. In the presence of the nutrient medium, the concentration of gibberellin (s) is usually between 0.1 mg / l and about 500 mg / l. Auxins can, for example, be used in a concentration of from 0.1 mg / l to 200 mg / l. Cytokinins can be used, for example in a concentration of from 0.1 mg / l to 100 mg / l.
    The nutrient medium may also include antimicrobials. Suitable antimicrobials are available from Sigma-Aldrich, St. Louis, Missouri, and sold as Product No. A5955. Antimicrobial agents can be used, for example, in a concentration of 1 ml / l.
    The methods of the invention may also be carried out with nutrient medium which includes a substance which renders the medium semi-solid or has a solidified consistency under normal ambient conditions. The nutrient medium may, for example, be in the form of a hydrated gel. A “gel” is a substance that is prepared as a colloidal solution and that forms or can be made to form a semi-solid material. Such a conversion of a liquid gel solution to a semi-solid material is referred to herein as "curing" or "solidification" of the gel. The term “hydrated gel” refers to a water-containing gel. Such gels are prepared by first dissolving in water (where the water acts as a solvent or "continuous phase") a hydrophilic polymeric substance (acting as a solute or "dispersed phase"), which on curing is combined with the continuous phase to form the semi-solid material. The water thus becomes homogeneously 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 the 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" when touched when the relative amount of water in the gel decreases.
    In addition to being water-soluble, suitable solutes for the gel are neither cytotoxic nor substantially phytotoxic. The term "substantially non-phytotoxic" substance as used herein 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 , destroy 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 hydrophilic 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 called “reversib | a”, because reheating a cured gel regenerates the gel solution. Solutions of other solutes in the gel require a “complex-forming” substance, which serves to chemically cure the gel by cross-linking 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 iron. 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 required for the preparation of a satisfactory gel according to the present invention varies depending on the particular solute 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. By practicing the methods of this aspect of the invention, the present inventors have provided a modified (improved) nutrient medium for use in manufactured seeds, as described in Example 2. As described in Examples 3 and 4, the present invention has inventors further discovered by experiment that a seed produced comprising a modified nutrient medium containing from 10 g / l to 100 g / l of a non-nutrient treated adsorbent material and from 350 mg / l to 450 mg / l NH 4 NO 3, from 2000 mg / l 1 to 3000 mg / l KH2PO4 and at least one component selected from the group consisting of from 150 mg / l to 300 mg / l myoinositol, from 1.5 mg / l to 3.0 mg / l thiamine-HCl, from 0.30 mg / l to 0.80 mg / l pyridoxine-HCl, from 1.5 mg / l to 3.0 mg / l nicotinic acid, from 0.15 mg / l to 0.30 mg / l ribo-avin, from 0.75 mg / l to 2.0 mg / l calcium pantothenate, from 0.01 mg / l to 0.03 mg / l biotin and from 0.15 mg / l to 0.30 mg / l folic acid, gives a improvement igroning the degree and normality of germs compared to a manufactured seed including a conventional nutrient medium (ie KE64) including 60 g / l nutrient-treated charcoal.
    In certain embodiments, the modified nutrient medium for use in the seed produced comprises about 60 g / l of non-nutrient treated charcoal, from about 350 mg / to about 375 mg / L NH 4 NO 3, from about 2000 mg / L to about 2100 mg / L KH 2 PO 4 and at least one component selected from the group consisting of about 200 mg / L myoinositol, about 2.0 mg / L thiamine HCl, about 0.50 mg / L pyridoxine HCl, about 2.0 mg / L nicotinic acid , about 0.26 mg / l riboflavine, about 1.0 mg / l calcium pantothenate, about 0.02 mg / l biotin and about 0.25 mg / l folic acid.
    In some embodiments, the seed produced also includes a shoot restriction, where the shoot restriction includes a cavity that is sized to receive the conifer embryo. In some embodiments, the seed produced also includes a coniferous embryo placed in the cavity of the shoot restriction.
    In some embodiments, the seed produced also includes an adsorbent material, such as charcoal, in the cavity. In some embodiments, the charcoal in the cavity is nutrient treated. In one example of an embodiment, the seed produced comprises a nutrient medium comprising about 60 g / l of non-nutrient treated charcoal, from about 350 mg / to about 375 mg / l NH 4 NO 3, from about 2000 mg / l to about 10 mg. G83 16 2100 mg / l KH2PO4 and at least one component selected from the group consisting of about 200 mg / l myoinositol, about 2.0 mg / l thiamine-HCl, about 0.50 mg / l pyridoxine-HCl, about 2.0 mg / l nicotinic acid, about 0.26 mg / l riboavine, about 1.0 mg / l calcium pantothenate, about 0.02 mg / l biotin and about 0.25 mg / l folic acid.
    The medium MSO9, described in Examples 1, 3 and 4, is an example of a modified nutrient medium for use in manufactured seeds and in the germination processes, as described herein.
    The modified (improved) nutrient medium formed by the methods of the invention and manufactured seeds including the modified nutrient medium can be used to germinate a conifer embryo. The method according to this aspect of the invention comprises (a) placing a conifer embryo in functional contact with a nutrient medium in a manufactured seed, the nutrient medium comprising from 10 g / l to 100 g / l charcoal from 350 mg / l to 450 mg / l NH4NO3, from 2000 mg / l to 3000 mg / l KH2PO4 and at least one component selected from the group consisting of from 150 mg / l to 300 mg / l myoinositol, from 1.5 mg / l to 3, 0 mg / l thiamine-HCl, from 0.30 mg / l to 0.80 mg / l pyridoxine-HCl, from 1.5 mg / l to 3.0 mg / l nicotinic acid, from 0.15 mg / l to 0.30 mg / l ribo-avin, from 0.75 mg / l to 2.0 mg / l calcium pantothenate, from 0.01 mg / l to 0.03 mg / l biotin and from 0.15 mg / l to 0, 30 mg / l folic acid; and (b) placing the produced seed in an environment leading to plant growth to enable the embryo to grow and germinate on the basis of the produced seed.
    As described above, the present inventors have experimentally discovered that a manufactured seed, including a modified nutrient medium, improves the germination rate of conifer embryos compared to a standard nutrient medium (eg KE64). The modified nutrient medium described herein in connection with the produced seeds is also useful in embryo germination procedures. In such embodiments of the process, the charcoal in the modified nutrient medium is not nutrient treated prior to addition to the medium. In some embodiments, the modified nutrient medium comprises from 10 g / l to 100 g / l charcoal. In certain embodiments, the charcoal added to the modified nutrient medium is from 10 g / l to 100 g / l of non-nutrient treated charcoal (such as from 20 g / l to 100 g / l, from 50 g / l to 100 g / l l, from 60 g / l to 100 g / l or from 50 g / l to 80 g / l, or about 60 g / l). In certain embodiments of the process, the modified nutrient medium for use in the seed produced comprises about 60 g / l of non-nutrient treated charcoal, from about 350 mg / to about 375 mg / l NH 4 NO 3, from about 2000 mg / l to about 2100 mg / l KH2PO4 and at least one component selected from the group consisting of about 200 mg / l myoinositol, about 2.0 mg / l thiamine-HCl, about 0.50 mg / l pyridoxine -HCl, about 2.0 mg / l nicotinic acid, about 0.26 mg / l riboflavine, about 1.0 mg / l calcium pantothenate, about 0.02 mg / l biotin and about 0.25 mg / l folic acid.
    The following examples illustrate only the best mode presently contemplated for the practice of the invention, but this should not be construed as limiting the invention.
    EXAMPLE 1 This example provides a representative process for the preparation of a suitable nutrient medium, nutrient-treated charcoal and representative manufactured seeds suitable for use in the processes of the invention.
    Methods: 1. Nutrient medium (KE64-50): 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 heat-labile chemicals and compounds are added, 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 1: FORMULATION OF THE BASIC MEDIA KE64 Final concentration 301 1 10 0 Medium grain 0 06 299 1800 0 1000 O 535 C183 1 8 Medium component Final concentration (mg / l) MnCl2-4H2O 6.0 ZnSO4-7H2O 0.8 CuCl2-ZHZO 0.5 Ferric citrate 60 mg / l Pluronic F-68 Qg / l Agar 18 g / l Filter-sterilized, heat-labile components (Table 2) are added after the medium has been cooled to 40 ° C.
    TABLE 2: Components added to the base medium KE64 Medium component Final concentration (mM) Final concentration (mg / I) Myoinositol 0.5549 100.0 Thiamine-HCl 0.0030 1.0 Pyridoxine-HCl 0.0012 0.25 Nicotinic acid 0.0081 1, Riboflavin 0.0021 0.125 Calcium 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-2HCl 0.7612 53.3 DL-serine 0.4631 80 L-proline 1.5310 53.3 L-arginine-HCl 0.4552 266.7 Urea 13.320O 800 L-valine 05983 53.3 L-alanine 0.2203 53.3 L-leucine 0.2448 80 L-threonine 0.3226 26.7 L-phenylalanine 0.172O 53.3 L-histidine 0.1308 26.7 L-tryplofan 0.2035 26.7 L-isoleucine 1.2930 26, 7 L-methionine 0.7100 26.7 Iglycine 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 Antimicrobials 1 .0 ml / I 10 15 20 25 30 535 G83 19 2. Preparation of charcoal for addition to the medium and / or to the corrosion resistance of the manufactured seeds A. Preparation of nutrient-treated charcoal: Base medium K E64 (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 in Table 2, described in Example 1, are added sterile to the base medium KE64, and the medium is stirred for at least 2 hours to mix the components. The agent is filtered through Whatman No. 1 filter paper in a Buchner funnel to collect the charcoal. A moisture balance path is used to determine the moisture content of the charcoal cake, and the dry weight of the charcoal is calculated. If the nutrient-provided charcoal is to be added to the integrity of the produced seed, it is first dried until it becomes a liquid material.
    B. Preparation of a Nutrient Treated Charcoal: 100 mesh charcoal chemically activated using a phosphoric acid process (NORIT® CNSP) was obtained from Norit Americas Inc., Marshall, Texas. 3. Manufacture of Manufactured Seeds: Representative methods used for the manufacture of manufactured seeds are described in U.S. Patent Nos. 6,119,395, 5,701,699 and 5,427,593, which are incorporated herein by reference.
    In general, manufactured seeds include a seed coat (24), a nutrient medium (26), a plant embryo (42) and optionally a heart leaf restriction (22). A manufactured seed that does not include a plant embryo (42) is known as a “seed blank.” The seed blank is usually a cylindrical capsule having a closed end and an open end.
    The nutrient medium (26), also referred to as "gametophyte medium", is analogous to the gametophyte of a natural seed and is placed in the seed coat for the purpose of substantially filling the interior of the seed coat. described KE64 or a modified nutrient medium, which as described herein may include from 0 g / l to 100 g / l of an adsorbent composition, such as charcoal. , as described above, or it may be pure, non-nutrient treated charcoal.
    A longitudinally extending hard porous insert, known as a cotyledon restriction (22) is centrally located at one end of the seed coat surrounded by the nutrient medium and includes a centrally located cavity (34), also called a "corrosion cavity", which partially extends through the length of the heart leaf restriction. The cavity (34) is sized to receive a plant embryo (42) therein. The well-known plant embryo includes a root end and a heart leaf end. The plant embryo is deposited in the holiness of the heart leaf restriction (22) with the heart leaf end first. The plant embryo is then closed in the seed by means of an end closure (43). A weakened point in the end closure (43) enables the root end of the plant embryo to penetrate the end closure.
    In an example of a process for producing a manufactured seed for use according to the invention, the seed coat is produced by cutting a polycaprolactone hose to a suitable length. Ceramic shot restraints are made by injecting a porcelain strip into a preformed template using a needle in the center to achieve the shot accepting holiness.
    The strip is allowed to dry to a consistency that allows removal of the preformed constraint. The boundary is then heated to a temperature which enables 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 stretching ParafilmW '(Pechiney Plastic Packaging, Chicago, Illinois 60631).
    The produced seed is assembled by thermobonding the ceramic shoot restriction (22) to the seed coat (24). The seed coat (24) is then filled with nutrient medium (26), and an embryo is inserted into the cavity (34) in the heart leaf boundary (22) with the heart leaf end first. Dry charcoal filler material (80) (either nutrient-treated or non-nutrient-treated) can be filled into the heart leaf restriction after the embryo is inserted into the cavity (34).
    After the charcoal has been added, the seeds are closed with a second end closure by placing it over the open end of the seed and fusing the lids to the surface by means of heat. The primary end closures are dipped in blue wax mixture before adhering to the second end closure. This promotes a satisfactory connection between the primary and secondary end connections. The seeds are then brushed with antimicrobial agents. 4. Preparation of plant embryos: 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 with the help of a scalpel and tweezers in a fume hood with a laminar fl fate.
    Somatic embryos are prepared using standard methods previously described (see, for example, 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 cultured 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 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 post-development treatments. 5. Germination: An appropriate amount of sterile sand is prepared by firing 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. Furrows are then created, and the box is closed. The box containing the 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. Usually, the seeds produced are grown in continuous light at room temperature (23 ° C) for four to five weeks. Several parameters can be measured to determine the germination frequency of the quality of the produced seeds and germs. At a fixed time after sowing, the lengths of the rootstock, hypocotylene, heart leaves and epicotylene of the germs can be measured.
    The term “root substance” refers to the part of a plant embryo that develops into the primary root of the resulting plant.
    The term "heart leaf" generally refers to the first, first pair or first wreath (depending on the type of plant) of leaf-like structures on the plant embryo which mainly serves to make nutrient compounds in the seed available to the developing embryo, but which in some cases function as nutrient storage 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 stem of the seedling which is located above the heart leaves.
    The degree of germination can be measured and the normality of the germs can also be assessed. A "normal germ" or "normality" denotes the presence of all the expected parts of a plant at the time of evaluation. In the case of gymnosperms, normality is characterized by the fact that the root substance has a length of more than 3 mm and no visible discernible malformations compared with the appearance of embryos that germinate on the basis of a natural seed.
    "Abnormal" means that tissue on at least one organ is swollen and that the root and heart leaves are dead. "Fully extracted but abnormal" means that the germ has completely pushed out of the cavity, but that it is not normal.
    "Unchanged" means that the embryo has not changed from day one of the experiment (ie no germination has taken place).
    EXAMPLE 2 This example describes the preparation of a modified nutrient medium for use in manufactured seeds containing non-nutrient treated charcoal.
    Methods: The KE64 medium, prepared as described in Example 1, was incubated in the presence or absence of nutrient-treated or non-nutrient-treated charcoal, and the concentration of the medium components was measured after incubation.
    The 100-mesh nutrient-treated charcoal was prepared as described in Example 1. 535,083 Tested conditions: 1. KE64 medium without added charcoal (sample 012). 2. KE64 medium plus 60 g / l non-nutrient-treated charcoal of 100 mesh (sample 013). 3. KE64 medium plus 60 g / l nutrient-treated 100 mesh charcoal (sample 014). Charcoal was added where indicated, mixed and incubated for 2 hours. The concentration of the medium components was analyzed as shown below in Table 2.
    TABLE 3: Comparison of measured concentration of medium components before and after incubation in the presence of charcoal. The measurement results are given in mg / I, unless otherwise stated.
    KE64 medium KE64 medium plus charcoal KE64 medium KE64 medium plus charcoal (nutrient (no charcoal) (no charcoal) (untreated) treated) Expected final- Measured Measured concentration concentration concentration concentration (mg / l) (mg / l l) (mgfl) (mg / l) Medium component “B” ”C” “D” ”E” NH4NOa 301.1 322.3 296.8 381.3 H3BO3 10.0 11.4 9.7 11.4 NH4 ) 2MoO4 0.06 0.08 0.02 0.02 CaCl2-2H2O 299.2 275.9 124.7 271.1 KH2PO4 1800.0 1573 2513 1907 MgSO4-7H2O 1000.0 987.3 635.6 960, MnClF4H2O 6.0 3.8 1.7 3.9 ZnSO4-7H2O 0.8 0.15 0.09 0.10 CuCl2-2H2O 0.5 0.27 <0.01 <0.01 Myoinositol 100.0 ND ND ND Thiamine-HCl 1.0 ND ND ND Pyridoxine-HCl 0.25 ND ND ND Nicotinic acid 1.0 ND ND ND Riboflavin 0.13 ND ND ND Calcium pantothenate 0.50 ND ND ND Biotin 0.0010 ND ND ND Folic acid 0.125O ND ND ND L-asparagine / serine 187 191 155 204 L-glutamine / histidine 293 231 166 206 535 083 24 KE64 medium KE64 medium plus charcoal KE64 medium KE64 medium plus charcoal (nutrient (no charcoal) ( no charcoal) (untreated) treated) Expected final- Measured Measured Measured concentration concentration concentration concentration (mg / I) (mg / I) (mg / I) (mg / I) Medium component “B” “C” “D” “E” L-lysine-HCl 53, 3 43 21 32 DL-serine 80.0 ND ND ND L-proline 53.3 94 87 100 L-arginine-HCl 266.7 256 43 76 L-valine 53.3 50 41 49 L-alanine 53.3 45 40 49 L-leucine 80.0 75 48 64 L-threonine 26.7 44 37 42 L-phenylalanine 53.3 51 5 10 L-tryptophan 26.7 19 ND ND L-isoleucine 26.7 25 16 22 L-methionine 26.7 18 ND ND glycine 53.3 52 40 51 L-tyrosine 53.3 56 3 6 L-cysteine 26.7 ND ND ND Sucrose 50.0 g / l 52.8 g / l 49.2 g / l 63.2 gll Urea 800 ND ND ND TABLE 4: Analysis of the results in TABLE 3 Increase in nutrient concentration in supernatant Concentration due to addition absorbed to of treated Adjustment charcoal, mg / I charcoal to factor for New starting medium, mg / l medium concentration (CD) (EC) (E / C) (HxW) Medium component “F” ”G” “H” ”l” NH4NO3 25.5 59.0 1.2 361.3 H3BO3 1.7 0.0 1.0 10.00 (NH4) 2MoO4 0.1 -0.1 1.0 0.1 caciz-zHzo 151, 1 -4.8 1.0 299.2 KHZPO., -940.3 333.9 1.1 205913 MgSO 4 -7H 2 O 351, 7 -26.4 1.0 1000.0 Mnciz-4Hzo 2.1 0.1 1.0 6.0 znso fl H o, 1 1.0 0.8 535 083 25 Increase in nutrient concentration in supernatant Concentration due to addition absorbed to of treated Adjustment charcoal, mg / I charcoal to factor for New starting medium, mg / I medium concentration (CD) (EC) (E / C) (HxB) Medium component “F” “G” “H” ”I” CuCl2-2H2O ND ND 1.0 0.5 Myoinositol ND ND ND 100.0 Thiamine-HCl ND ND ND ^ 1 .0 Pyridoxine-HCl ND ND ND 0.3 Nicotinic acid ND ND ND 1.0 Riboflavin ND ND ND 0.1 Calcium pantothenate ND ND ND 0.5 Biotin ND ND ND 0.0 Folic acid ND ND ND 0.1 L-asparagine / serine 36.0 13.0 1.1 L-asparagine: 117.4 Serine: 88.0 L-Gutamine / histidine 65.0 -25.0 1.0 L-Gutamine: 266.7 L-histidine: 26, 7 L-lysine-HCl 22.0 -25.0 1.0 53.3 L-roline 7.0 6.0 1.1 58.6 L-arginine-HCl 213.0 -180.0 2.0 533 , 4 L-valine 9.0 -1.0 1.0 53.3 L-alanine 5.0 4.0 1.1 58.6 L-leucine 27.0 -11.0 1.0 80.0 L -treonine 7.0 -2.0 1.0 26.7 L-phenylalanine 46.0 -41.0 1.0 53.3 L-isoleucine 9.0 -3.0 1.0 26.7 L-glycine 12.0 -1.0 1.0 53.3 L-tyrosine 53.0 -50.0 1.0 53.3 L-cysteine ND ND ND 53.3 Sucrose 3.6 10.4 1.2 60.0 Urea ND ND 1.0 800.0 535 G83 26 TABLE 5: Modified nutrient medium MS08 and MS09 KE64 medium MS08 MS09 Medium component Final concentration Final concentration Final concentration (mg / l ) (mg / U (mg / l) NH4NO3 301.1 301.1 371.7 H3BO3 10.0 10.0 10.0 (NH4) 2MoO4 0.06 0.06 0.06 CaCl2-ZHZO 299.2 299 .2 299.2 KH2PO4 1800.0 2088 2088 MgSO4-7H2O 1000.0 1000 1000 MnCl2-4H2O 6.0 6.0 6.0 ZnSO4 ~ 7H2O 0.8 0.8 0.8 CuCl2-ZHZO 0.5 0 .5 0.5 Iron citrate 60 mg / I 60 60 Myoinositol 100 100 200 Thiamine-HCl 1.0 1.0 2.0 Pyridoxine-HCl 0.25 0.25 0.50 Nicotinic acid 1.0 1.0 2.0 Riboflavin 0.125 0.13 0.26 Calcium pantothenate 0.50 0.50 1.0 Biotin 0.0010 0.01 0.02 Folic acid 0.1250 0.13 0.25 L-asparagine 106.7 11.73 11.73 L-glutamine 266.7 266.7 266.7 L-lysine-2HC | 53.3 53.3 53.3 DL-serine 80.0 88.0 88.0 L-proline 53.3 58.63 58.63 L-arginine-HCl 266.7 533.3 533.3 Urea 800, 0 800 800 L-valine 53.3 53.3 53.3 L-alanine 53.3 58.63 58.63 L-leucine 80.0 80.0 80.0 L-threonine 26.7 26.7 26, 7 L-phenylalanine 53.3 53.3 53.3 L-histidine 26.7 26.7 26.7 L-tryptophan 26.7 26.7 26.7 L-isoleucine 26.7 26.7 26.7 L -methionine 26.7 26.7 26.7 L-glycine 53.3 53.3 53.3 L-tyrosine 53.3 53.3 53.3 L-cysteine 26.7 26.7 26.7 Pluronic F- 68 10 g / l 9.0 gll 9.03 !! 535 C183 27 KE64 medium MS08 MS09 Medium component Final concentration Final concentration Final concentration (mg / I) (mg / I) (mg / I) Charcoal 60 g / l 60.0 g / l 60.0 g / l (nutrient (non-nutrient) nes- (not nutrient- treated) treated) treated) _A_tgar 183 - 26 g / l 18.0 g / l 18, (Lg / l pH 5.7 5.7 Sucrose 50 g / l 60.0 g / l 60 .0 g / l TABLE 6: Summary of components that differ in modified MS-08 and MS-09 medium compared to KE64 KE64 medium MS08 MS09 Medium component Final concentration Final concentration Final concentration (mg / I) (mg / I) (mg / I) NH4NO3 301.1 301.1 371.7 KH2PO4 1800.0 2088 2088 Myoinositol 100 100 200 Thiamine-HCl 1.0 1.0 2.0 Pyridoxine-HCl 0.25 0.25 0.50 Nicotinic acid 1, 0 1.0 2.0 Riboflavin 0.125 0.13 0.26 Calcium pantothenate 0.50 0.50 1.0 Biotin 0.0010 0.01 0.02 Folic acid 0.1250 0.13 0.25 L-asparagine 106 .7 11.73 11.73 DL-serine 80.0 88.0 88.0 L-proline 53.3 58.63 58.63 L-arginine-HCl 266.7 533.3 533.3 L-alanine 53 .3 58.63 58.63 Charcoal 60 g / l 60.0 g / l 60.0 g / l (nutrient- (not nutritious) nutrient- (non-nutrient- treated) treated) treated) _Agar 18 g - 26 g / l 18.0 g / l 18.0 g / l H 5.7 5.7 Sucrose 50 g / l 60.0 g / l 60.0 g / l It can be noted that the concentration of some of the components, such as L-tyrosine, L-phenylalanine and ferric citrate, was adjusted with respect to the pH value and to avoid precipitation problems. 10 15 20 25 30 535 G83 28 EXAMPLE 3 This example describes a comparison of the effect of different nutrient formulations of type KE64, MS08 and MS09 used in the manufactured seed on the germination frequency and quality of yellow southern state stables.
    Methods: Zygotic seeds of yellow southern state pine ("Loblol | y pine") were surface sterilized, and embryos were removed and inserted into the produced seed, as described in Example 1.
    Manufactured seeds were prepared as described in Examples 1 and 2 using ceramic leaf blade constraints and with the variations of nutrient medium and either nutrient-treated or non-nutrient-treated charcoal included in the cavity shown below. The nutrient medium KE64 was prepared as described in Example 1. Nutrient treated charcoal was prepared as described in Example 1. The MS08 and MS09 media were prepared as described in Example 2.
    Once the manufactured seeds were assembled for the indicated constructions, dry nutrient-treated charcoal or non-nutrient-treated charcoal of 100 mesh was then pipetted into the corrosion cavity of the structure using a sterile pasteur pipette. The embryos were then inserted into the heart leaf restriction.
    The nutrient medium was prepared as shown below and placed in the seed coat so that it substantially filled the interior of the seed coat. Under certain treatment conditions, the nutrient medium contained charcoal that was nutrient-treated, and under other treatment conditions, the medium contained charcoal that was not nutrient-treated.
    As described above in Example 2, MS08 and MS09 were formulated to increase the concentration of certain medium components, as shown in Tables 5 and 6, compared to KE64 in order to try to improve, or at least maintain, the same germination frequency as observed with KE64. in the presence of nutrient-treated charcoal.
    The medium dispersion temperature was 45 ° C. The living end of the produced seed (the end with the embryonic cavity) was dipped in wax. 10 15 20 535 G33 29 108 seeds were tested per treatment (4 treatments), resulting in a total of 432 seeds.
    Tested treatment conditions: 1.
    Complete KE64 medium (50 g / l sucrose; 18 g / l agar) plus 60 g / l nutrient-treated charcoal in the medium and nutrient-treated charcoal in the whole.
    Complete MS08 medium (60 g / l sucrose; 18 g / l agar) plus 60 g / l non-nutrient-treated charcoal in the medium and nutrient-treated charcoal in the cavity.
    Complete MS09 medium (60 g / l sucrose; 18 g / l agar) plus 60 g / l non-nutrient-treated charcoal in the medium and nutrient-treated charcoal in the whole.
    Complete KE64 medium (50 g / l sucrose; 18 g / l agar) plus 60 g / l non-nutrient-treated charcoal in the medium and nutrient-treated charcoal in the whole.
    Produced seeds composed as described above for each treatment condition were sown in boxes of sterile sand and placed in a bright room. The seeds were recorded for germination 25 days after sowing.
    Results: The results are shown in Tables 7 and 8 below.
    TABLE 7: Or lengths and% lateral deviations ("laterals") Side deviations- Root substance- Hypocotyl- Heart leaf- Epicotyl- Medium deviations length length length length (%) (mm) (mm) (mm) (mm) Treatment no. 1 25.3 % 27.0 22.7 19.5 8.2 (KE64 plus 60 g / l nutrient-treated charcoal in the medium) Treatment no. 2 20.4% 27.0 23.3 21.2 2.6 (MS08 plus 60 g / l non-nutrient-treated charcoal in the medium) Treatment no. 3 43.0% 28.9 28.1 20.5 9.5 (MS09 plus 60 g / l non-nutrient-treated charcoal in the medium) 535 G83 30 Heart leaf- Epicotyl- Medium folds length length length length (%) (mm) (mm) (mm) (mm) Treatment no4 4.3% 10.9 14.6 16.4 '4.7 (KE64 plus 60 g / l not nutrient-treated charcoal in the medium) TABLE 8: Seedling quality Would be normal if Total share Medium Normal they would be full- normal Abnormal sprout- constantly sprout- sprout- Raw materials extracted substances substances changed (° / °) (%) (column 1 + column 2) (%) (%) Treatment No. 1 30.3% 5.1% 35.4% 38.4% 26.3% (KE64 plus 60 g / l nutrient b processed charcoal in the medium) Treatment no. 2 22.1% 2.1% 24.2% 32.6% 43.2% (MS08 plus 60 g / l non-nutrient-treated charcoal in the medium) Treatment no. 3 44.7% 10, 6% 55.3% 26.6% 18.1% (MS09 plus 60 g / l non-nutrient-treated charcoal in the medium) Treatment no. 4 3.2% 2.1% 5.3% 43.6% 51.1% ( KE64 plus 60 g / l non-nutrient-treated charcoal in the medium) Result discussion: As shown above in Table 8, the produced seeds with MS09 with 60 g / l non-nutrient-treated charcoal in the medium gave better results than KE64 with 60 g / l nutrient-treated charcoal in the medium, the KE64-containing seeds resulted in a germination frequency of 44.7% in normal germs and a total frequency in normal and normal, but not fully extracted, germs of 55.3% compared to the MS09-containing seeds, which gave 30.3% normal germination frequency, and a total germination frequency of 10 15 20 25 30 535 D83 31 35.4% for normal and normal, but not completely extracted, germs.
    The MS08 medium with 60 g / l non-nutrient treated medium did not give as good results as KE64 or MS09.
    In addition, as shown in Table 7, the seeds produced with MS09 medium (with non-nutrient treated charcoal in the medium) yielded germs with organ sizes at least as large, if not larger, than those of the germs produced from the standard KE64 medium. the medium (with nutrient-treated charcoal in the medium).
    Overall conclusion: It has been shown that MS09 is superior to both KE64 and MS08 for use in manufactured seeds of yellow southern state stables with respect to the frequency and quality of the resulting germs. The use of this modified medium (MS09) provides the advantage of avoiding the time and cost involved in the prolonged preparation of nutrient treated charcoal for use in manufactured seeds.
    It can be noted that a further experiment was performed with embryos of yellow southern state pine for comparison of produced seeds containing either KE64 with 60 g / l non-nutrient-treated charcoal or MS09 with 60 g / l non-nutrient-treated charcoal, the germination results being assessed 49 days after sowing. but this experiment did not yield any statistically significant data, probably due to contamination problems.
    EXAMPLE 4 This example describes the effect of different nutrient media used in manufactured seeds on the germination frequency and quality of Douglas fir embryos.
    Methods: Douglas fir somatic embryos from two different genotypes (genotypes # 1 and # 2) were grown up to the development stage, as described in Example 1.
    These embryos were then placed on a stratification medium for 4 weeks and evaluated for sterility before insertion into manufactured seeds. Manufactured seeds were prepared as described in Example 1 using either Type A ceramic leaf blade restrictions or Type B ceramic leaf blade restrictions and with the variations in nutrient medium and either nutrient-treated or non-nutrient-treated charcoal listed below. included in the cavity. The nutrient medium KE64 was prepared as described in Example 1. Nutrient treated charcoal was prepared as described in Example 1. The MSO9 medium was prepared as described in Example 2.
    Once the fabricated seed samples were assembled for the indicated constructs, dry, nutrient-treated charcoal or 100-mesh nutrient-treated charcoal was then pipetted into the corrosion cavity of the structure using a sterile pasteur pipette. The embryos were then inserted into the heart leaf restriction.
    The nutrient medium was prepared as shown below and placed in the seed coat to substantially fill the interior of the seed coat. Under certain treatment conditions, the nutrient medium contained charcoal that was nutrient-treated, and under other treatment conditions, the medium contained charcoal that was not nutrient-treated.
    As described above in Example 2, MSO9 was formulated to increase the concentration of certain medium components, as shown in Tables 5 and 6, compared to KE64 in an attempt to increase, or at least maintain, the same germination rate observed with KE64 in the presence of nutrient treated charcoal.
    The medium dispersion temperature was 45 ° C. The living end of the produced seed (the end with the embryonic cavity) was dipped in wax.
    Each treatment in this study consisted of 7 copies with 10 seed copy treatment, ie a total of 280 seeds.
    The treatment conditions are described in Table 9 below. The produced seeds containing embryos were sown in 7 sterile sandboxes with 10 seed treatment / box. The seeds were registered with respect to germination frequency and organ lengths 61 days after sowing. 535 B83 33 TABLE 9: Treatment conditions for the production of produced seeds using genotypes No. 1 and No. 2 of somatic embryos of Douglas fir Treated- Tested Nutrient Charcoal in Nutrient Charcoal in cavity Heartbeat genotypes medium medium 1 No. 1 KE64 nutrient treatment- nutrient- Type B No. 2 lat (60 g / l) treated charcoal in cavity 2 No. 1 MS09 not nutrient-nutrient- Type B treated charcoal treated charcoal in (60 g / l) cavity 3 No. 1 KE64 nutrient treatment nutrient - Type B no. 2 lat charcoal (60 g / l) treated charcoal in lÉQhet 4 no. 2 MS09 not nutrient nutrient- Type B treated charcoal (60 g / l) treated charcoal in cavity 5 no. 1 KE64 nutrient treated charcoal in Type A lat charcoal (60 g / l) cavity 6 no. 2 KE64 nutrient-treated charcoal in Type A lat charcoal (60 g / l) cavity TABLE 10: Organ lengths for all treatments (mm) Rotäm nes- Hypocotyl- Heart leaf- Epicotyl- length (mm) length (mm) length (mm) length (mm) Treat d ng d = 0.0014 a = 0.0001 a = 0.0001 a = 0. 6717 Nr1 9.51 mmB 1o, 46 ° 5.348 0.00 (geno nr 1 / nr 2: KE64: treated charcoal in medium and cavity No. 2 12.89 mm ^ ß 13.00 mm ° 7.24 mm ^ W 7.75 mm (geno No. 1: MS09: untreated charcoal in medium, treated charcoal in cavity) No. 3 5.93 mmß 11.26 mm "5.15 mm" 5.40 mm (geno no. 1 / no. 2: KE64: treated charcoal in medium, treated charcoal in cavity) No. 4 7.81 mms 10.47 mmBC 5.15 mmB 2.60 mm ( geno no. 2: MS09: untreated charcoal in medium, treated charcoal in cavity) 535 D83 34 Root substance- Hypocotyl- Heart leaf- Epicotyl- length (mm) length (mm) length (mm) length (mm) Treatment G = 0.0014 C1 = 0.0001 G = 0.0001 Cl = 0.6717 No. 5 15.71 mm ”14.76 mm ^ 9.57 mm ^ 3.51 mm (geno No. 1: KE64: treated charcoal in medium, no charcoal i _ @ ghet No. 6 23.04 mm ^ 17.45 mm ^ 9.03 mm ^ 3.52 mm (geno No. 2: KE64: treated charcoal in medium, no charcoal in cavity TABLE 11: Germination frequency of Douglas fir Full Partial Total No germination germination germination Root in air germination Treatment d = 0.00131 or = 0 .0087 (col 1 + 2) d = 0.4614 d = 0.0011 Nr1 o, o% B 111% ”11.1% 11.1% 75.9% ^ (geno nr 1 / nr 2: KE64: treated charcoal in medium and cavity) No. 2 1.9% ”259%” 27, s% 7.4% 64, s% ^ B (geno No. 1: MS09: untreated charcoal in medium, treated charcoal in hollow fi t) No. 3 90% * 56% ”5.6% 167% 77.6% ^ (geno no. 1 / no. 2: KE64: treated charcoal in medium, treated charcoal in cavity No. 4 90%” 165% ”165% 146% 667 % ”(Geno no. 2: MS09: untreated charcoal in medium, treated charcoal in cavity) No. 5 11.1% ^ 222%” 333% 195% 4s, 1% ß (geno no. 1: KE64: treated charcoal in medium , no charcoal in cavity) No. 6 93% ”29.6% ^ 369% 9.3% 4s, 1% B (geno No. 2: KE64: treated charcoal in medium, no charcoal in cavity) 1Average followed by the same letter no significantly different. zRot in air is a negative result and indicates that the root has lost its geotropism. 535 ÜBíÉ 35 TABLE 12: Normalization entries for all treatments Would be normal if it were Total Unchanged- complete germination row (none Normal extracted (columns germination) Abnormal Treatment or = 0, O0011 u = 0.0087 1 + 2) oi = 0.0273 oi = 0.1174 No. 1 0.0% ° 13.0% 13.0% 38.9% 37.0% (geno No. 1 / No. 2: KE64: treated charcoal in medium and cavity) No. 2 9.3% B ° 259% 352% 213% 352% (geno No. 1: MS09: untreated charcoal in medium, treated charcoal in cavity) No. 3 1.9% ° 93% 112% 33 .3% 556% (geno no. 1 / no. 2: KE64: treated charcoal in medium, treated charcoal in cavity) Nr4 1.9 ° / °° 18.5% 20.4% 31.5% 42 .6% (geno no. 2: MSO9: untreated charcoal in medium, treated charcoal in cavity) No. 5 1e, 7 ° / ° ^ 222% 339% 463% 143% (geno no. 1: KE64: be - treated charcoal in medium, no charcoal in cavity) No. 6 25.9% ^ 22.2% 48.1% 33.3% 16.7% (geno no. 2: KE64: treated charcoal in medium, no charcoal in cavity) 1Average followed by the same letter not significantly different.
    Discussion of results: As shown above in Tables 10-12, there is an increase in germination frequency and organ size of germs from manufactured seeds containing the modified nutrient medium MS09 with non-nutrient treated medium compared to manufactured seeds containing KE64 medium with nutrient treated medium. The coating of charcoal with nutrients is a complex and time consuming process. The use of the modified MS09 medium with non-nutrient-treated medium therefore provides a significant advantage in the production of manufactured seeds and methods for germinating plant embryos. 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 (20)
    [1]
    A method of preparing an improved nutrient medium comprising an adsorbent material for growing plant cells, the method comprising: (a) determining whether there is a reduction in the concentration of one or fl your components in a first nutrient medium after incubation with a desired amount of adsorbent material; and (b) preparing an improved nutrient medium comprising the same components as the first nutrient medium, the improved nutrient medium comprising: (i) an increased concentration of said one or more components which in step (a) were determined to have a reduced concentration in the presence of the adsorbent material; and (ii) the same type of adsorbent material within a concentration range that is at most twice as high as that used in step (a).
    [2]
    The method of claim 1, wherein the adsorbent material is selected from the group consisting of charcoal, polyvinylpyrrolidone, and silica gel.
    [3]
    The method of claim 1, wherein the adsorbent material is charcoal.
    [4]
    The method of claim 3, wherein the concentration of charcoal to be added to the improved nutrient medium is from about 1 g / l to about 100 g / l.
    [5]
    The method of claim 3, wherein the charcoal is not nutrient treated prior to addition to the nutrient medium.
    [6]
    A method according to claim 3, wherein the nutrient medium is intended for growth of coniferous cells.
    [7]
    The method of claim 1, wherein the improved nutrient medium comprises at least 2 components selected from the group consisting of NH 4 NO 3, MgSO 4, FeSO 4, myoinositol, thiamine-HCl, pyridoxine-HCl, nicotinic acid, ribovin, calcium pantothenate, biotin and folic acid, DL-serine, L-proline, L-arginine-HCl and L-alanine.
    [8]
    The method of claim 1, further comprising placing the first nutrient medium from step (a) in a first set of manufactured seeds and placing the improved nutrient medium from step (b) in a second set of manufactured seeds, placing of a conifer embryo in functional contact with the nutrient medium in each of the produced seeds from the first and second sets of produced seeds, placement of the produced seeds in an environment leading to plant growth and comparison of the germination frequencies of the embryos from the first and second sets of manufactured seeds.
    [9]
    A process for the preparation of an improved nutrient medium comprising an adsorbent material for culturing plant cells, the process comprising: (a) incubating a first nutrient medium comprising a predetermined initial concentration of components comprising one or fl carbon sources, vitamins, minerals and amino acids with a desired amount of adsorbent material for addition to an improved nutrient medium; (b) determining whether there is a reduction in the concentration of one or more of the components of the first nutrient medium after the incubation in step (a) compared to the predetermined initial concentration of the component; and (c) preparing an improved nutrient medium comprising the same components as the first nutrient medium, the improved nutrient medium comprising: (i) an increased concentration of said one or more components determined in step (b) to have a reduced concentration in the presence of the adsorbent material; and (ii) the same type of adsorbent material in a concentration range that is at most twice as high as that used in step (a). 10 15 20 25 30 535 G83 39
    [10]
    The method of claim 9, wherein the first nutrient medium is incubated with the adsorbent material for a period of at least 10 minutes up to one week.
    [11]
    The method of claim 9, wherein the adsorbent material is selected from the group consisting of charcoal, polyvinylpolypyrrolidone, and silica gel.
    [12]
    The method of claim 9, wherein the adsorbent material is charcoal.
    [13]
    The method of claim 12, wherein the concentration of charcoal to be added to the improved nutrient medium is from 1 g / l to about 100 g / l.
    [14]
    The method of claim 12, wherein the charcoal is not nutrient treated prior to addition to the nutrient medium.
    [15]
    A method according to claim 9, wherein the nutrient medium is intended for growth of coniferous celery.
    [16]
    The method of claim 9, wherein the nutrient medium comprises at least two components selected from the group consisting of NH 4 NO 3, KH 2 PO 4, MgSO 4, FeSO 4, myoinositol, thiamine-HCl, pyridoxine-HCl, nicotinic acid, riboflavin, calcium pantothenate, biciotin pantothenate folic acid, DL-serine, L-proline, L-arginine-HCl and L-alanine.
    [17]
    The method of claim 9, wherein the volume of the nutrient medium incubated in step (a) is 1/4 to 1/1000 of the volume of the improved nutrient medium from step (c).
    [18]
    The method of claim 9, further comprising placing the improved nutrient medium from step (c) in one or fl of your manufactured seeds.
    [19]
    The method of claim 18, further comprising placing a conifer embryo in functional contact with the nutrient medium in the manufactured seed. 10 535 ÜBB 40
    [20]
    The method of claim 9, further comprising placing the first nutrient medium from step (a) in a first set of manufactured seeds and placing the improved nutrient medium from step (b) in a second set of manufactured seeds, placing a conifer embryos in functional contact with the nutrient medium in each of the produced seeds from the first and second sets of produced seeds, placement of the produced seeds in an environment leading to plant growth and comparison of the germination frequencies of the embryos from the first and the second set of manufactured seeds.
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    法律状态:
    2018-05-29| NUG| Patent has lapsed|
    优先权:
    申请号 | 申请日 | 专利标题
    US24736409P| true| 2009-09-30|2009-09-30|
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