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    • 专利摘要:
    The present invention provides methods of removing liquid from a porous substrate on which plant embryos are disposed.
    公开号:SE1151290A1
    申请号:SE1151290
    申请日:2011-12-29
    公开日:2012-07-01
    发明作者:Patrick M Brownell；Robert A Starr；Ramon C Dezutter
    申请人:Weyerhaeuser Nr Co；
    IPC主号:
    专利说明:

    AWAPATENT ABWEYERHAEUSER NR COMPANY Office / HandlerAnsokningsnrVar. reference Malmo / Dan Henriksson / DHSE-21055153 1 PROCEDURE FOR REPRODUCING WATER FROM A POROST SUBSTRATE IN SOMATIC PLANT EMBRYOGENES Background Modern silver culture often requires planting of a large number of genetically identical plants that have been selected to have beneficial properties. Production of new plants through sexual reproduction, which yields botanical Mon, is usually not feasible. Asexual reproduction, through the cultivation of somatic or zygotic embryos, has been shown for some species to produce a large number of genetically identical embryos, each with the potential to develop into a normal growth.
    Somatic cloning is a process for producing genetically identical plants based on the flange of other plant tissue than male and female diameters. In a somatic cloning approach, growth tissue is grown in an initiation medium that includes hormones, such as auxins and / or cytokinins, to initiate the formation of embryogenic tissue, such as embryogenic suspensory masses, with the potential to develop into somatic embryos. The embryogenic tissue is then further cultured in a propagation medium that promotes the establishment and proliferation of the embryogenic tissue to form pre-cardiac embryos (ie, embryos that lack heartbeat). These pre-cardiac embryos are then grown in a development medium that promotes the development and maturation of cardiac somatic embryos which can, for example, be placed on a germination medium for the production of the sprout and then transferred to soil for further growth, or alternatively placed in manufactured irons and sown in soil, where they grow to obtain seedlings. Manufactured tons are described, for example, in U.S. Patent Nos. 5,564,224, 5,687,504, 5,701,699 and 6,119,395.
    The somatic embryogenesis process is usually labor intensive and inefficient. One of the steps in the process involves, for example, the transfer of embryos woven from liquid propagating media and subsequent spreading below low density on a semi-solid medium surface for embryo development and maturation. This step is usually performed manually by a person skilled in the art by using a pipette to disperse a mixture of embryogenic cells and a liquid medium on the development medium.
    Another laborious step in the embryogenesis process is the selective harvesting of the development medium of individual embryos suitable for germination. At the end of the development phase, the embryos can occur in a number of stages of maturation and development. Those that are most likely to grow into normal plants are preferably selected using a number of visually evaluated selection criteria, such as embryo size (shape, eg axial symmetry), heart leaf development, surface texture, color, etc., and are manually picked out using a developmental aid. . The selected Onskvarda embryos are then carefully laid out and separated from each other for further processing. This is a skilled but laborious job, which is time consuming and expensive. In addition, it results in a significant production bottleneck when the final desired production is in the millions of plants.
    Efforts have been made to automate the somatic embryogenesis process. Upscaling and automation of somatic embryogenesis technology may involve the use of large volumes of liquid media or water for dilution purposes and / or the singularization of immature and mature embryos for the purpose of moving and positioning the embryos for subsequent processing steps. Suspension cultures at the end of the propagation stage can, for example, be diluted in order to facilitate even smearing of the pre-cardiac embryos on a development medium.
    Another example of using large volumes of liquid is in the singularization step. Singularization is a processing step that occurs at the end of the developmental and maturation stage, when embryos are physically separated from each other and the underlying embryogenic suspensory mass (ESM) for further processing, such as insertion into manufactured seed or placement on a 3 germination or pre-germination medium. treatment before insertion into manufactured seeds. The singularization can be accomplished by spraying the embryos and associated ESM with fluid to remove them from the development medium, using a series of sieves to separate the embryos from each other and the remaining ESM, placing the embryos in large volumes of fluid, and then placing individual embryos. on a por6st substrate.
    The presence of excess fluid on the substrate on which the embryos spread during the smear step and / or the singularization step can be problematic. Avoiding excess moisture and retention of hormone residues in the liquid medium on the gel cell spruce surface is critical for the quality of embryonic development. The presence of fluid on the substrate on which the embryos are spread can also have significant negative effects on germination.
    Procedures are needed to remove the liquid surface of the embryos and the substrate on which the embryos are spread, without damaging the embryos or disturbing the position of the embryos on the substrate. The present invention is directed to these and other needs.
    SUMMARY The present invention provides a process for depositing the liquid from a porous substrate on which plant embryos are spread. The methods of the invention include the steps of (a) providing a porous substrate having a top surface and a bottom surface; (b) spreading growth embryos on the top surface of the porous substrate; (c) providing an inlet port in communication with a vacuum cold, the cross-sectional area of the inlet port being smaller than the bottom surface area of the porous substrate; (d) the step of bringing the inlet port and a portion of the bottom surface of the porous substrate with plant embryos spread on the corresponding top surface in close proximity to each other when the inlet port communicates with the vacuum source, a vacuum being applied to the portion of the bottom surface of the porous substrate 4 in the immediate vicinity of the inlet opening; and (e) moving the inlet port and the bottom surface of the porous substrate relative to each other while the inlet port communicates with the vacuum source until substantially the entire of a desired area of the bottom surface of the porous substrate has been in the immediate vicinity of the inlet port, thereby removing the liquid from the area of the porous substrate on which the plant embryos are spread.
    Description of the Drawings The above aspect 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 schematically shows an example of a vacuum system for use according to an embodiment of the methods according to the invention.
    Figure 2 schematically shows an embodiment of the methods according to the invention, in which a porous substrate with embryos spread on the top surface of the porous substrate is moved across an orifice in a vacuum housing.
    Detailed Description The term "embryogenic suspender mass" (ESM) refers to embryos at an early stage of the process of proliferation by budding and cleavage.
    It has used the term "embryogenic tissue" to refer to an aggregate of tens to hundreds of embryogenic cells which form an embryogenic suspension mass.
    It uses the term "plant embryo" to refer to either a zygotic plant embryo or a somatic plant embryo. A zygotic plant embryo is an embryo son found inside a botanical seed produced through sexual reproduction. Somatic growth embryos can be prepared 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. It has the term "plant embryo" including embryos at various stages of development and includes both pre-cardiac and cardiac embryos.
    It has used the term "pre-cardiac embryo" to refer to an embryo that does not yet have any cardiac leaves.
    It uses the term "cardiac embryo" to refer to an embryo having one or more cardiac leaves.
    It has used the term "liquid" to refer to any liquid which is used in the embryogenesis process including, but not limited to, water, isotonic solution or culture medium.
    It has used the term "smearing" to refer to procedures for spreading embryogenic suspension masses and / or embryos on a surface.
    It has used the term "singularization" in the process of separating cardiac embryos from one embryogenic suspension mass and other Iranian embryos to obtain individual embryos.
    The somatic embryogenesis process is a process for the development of plant embryos in vitro. Methods for producing somatic plant embryos are known in the art and have been previously described (see, for example, U.S. Patent Nos. 4,957,866, 034,326, 5,036,007, 5,041,382, 5,236,841, 5,294,549, 482,857, 563,061. and 5,821,126). The somatic embryogenesis process includes the steps of (1) initiating or inducing to initiate the formation of embryogenic tissue, such as embryogenic suspensory mass (ESM), which is a white mucous mass that includes embryos at an early stage with a long thin-walled suspender associated with a small head with tat cytoplasm and large nuclei; (2) proliferation, sometimes referred to as sustaining, for the establishment and proliferation of embryogenic tissue for the formation of pre-cardiac embryos, which may be characterized as having even embryonic heads, with a plurality of suspensions; (3) development, for the purpose of developing and forming mature heart-bladed sonatic embryos, and (4) post-development stages, such as singularization, stratification, germination, placement in manufactured irons and transfer to soil for further growth and development.
    As described above in the "Background" section, the somatic embryogenesis process is labor intensive. Efforts have been made to automate and scale up the process to facilitate the production of tens of thousands of plant embryos. The propagation step can, for example, be carried out in a liquid bioreactor on a commercial scale. At the end of the reproduction stage, pre-cardiac embryos can be transferred to the development medium.
    A method of transferring pre-cardiac embryos to a development medium is described in U.S. Patent No. 7,785,884. The transfer step may be performed, for example, by removing a volume of the suspension culture from a bioreactor, the step of letting the cells settle, and feeding the sedimented cell volume; diluting the sedimented cell volume with sterile dilution needles; uniform dispersion of the cells and the dilution medium with an undesired density on a porous substrate applied to a non-porous surface; removing the sterile dilution medium from the porous substrate, thereby enclosing the uniformly dispersed pre-leaf embryos on the porous substrate; and transferring the porous substrate with scattered pre-cardiac embryos to the developing medium.
    The sterile dilution medium can be removed from the porous substrate by a variety of methods. The porous substrate can, for example, be attached to a smear frame including handles and can be lifted vertically by means of the handles by means of any ground-lifting means, such as manually or by means of robotic devices. The sterile dilution medium 7 can also be removed by using any method which avoids disturbing the distribution of the spread cells, such as sucking, draining, tipping or leaching the sterile dilution medium.
    The methods described above are labor intensive and involve the transfer of porous substrate and scattered cells to multiple surfaces which are used only once or which need to be handled frequently to be ready for use once more.
    After smearing and deposition of fluid, the pre-cardiac embryos can be placed on a development medium for a period of time for development into cardiac embryos. At the end of the development period, the heart-leafed embryos are bound to and embedded in suspender tissues to varying degrees and remaining underdeveloped ESM, along with incompletely developed embryos, abnormally shaped embryos, undersized or oversized embryos and other parts of non-embryonic material and other embryos. It is important for subsequent normal germination to separate the embryos from the suspensory mass and from other embryos in order to obtain individual embryos. This separation process is called "singularization". Like the smearing process, the singularization is labor intensive. The embryos are usually selected by hand and transferred to a dry filter paper or medium using tweezers.
    Automation of the singularization step is important for commercial upscaling of the embryogenesis process, as well as for productivity and staff choice. In automated singularization, the embryos can be washed away from a development medium using an aqueous fluid, such as water or an isotonic nutrient solution, and passed through a series of sieves. During screening, the embryos can be further sprayed with aqueous liquid to facilitate removal and dewatering of any unwanted material, such as undersized embryos, tissues and residual embryogenic suspension masses. The singularized individual embryos can then be placed on a porous substrate for further processing.
    At the end of the automated singularization process, both the embryos and the porous substrate have free liquid on their surfaces. It is important to remove residual fluid from contact with the embryos, as fluid in contact with the embryos can have pronounced detrimental effects on the embryo's osmolality and water potential. If fluid is left in contact with the embryo, the resulting change in the embryo's water potential can, for example, result in undesired premature germination and extension.
    As described above, it is important in both the stage of smearing pre-cardiac embryos on the development medium and the stage of singularizing cardiac embryos to remove free fluid from the surface of the dispersed embryos and from the porous substrate on which the embryos are distributed. The present inventors have discovered methods for removing liquid from the surface of plant embryos and a porous substrate on which the plant embryos are spread, which results in a more complete and durable removal of liquid compared to other methods in the prior art (e.g. use of a Buchner funnel, suction , drainage, welding, etc.).
    The present invention provides methods for removing the liquid from a porous substrate on which the plant embryos are spread. The methods of the invention include the steps of (a) providing a porous substrate having a top surface and a bottom surface; (b) spreading growth embryos on the top surface of the porous substrate; (c) providing an inlet port in communication with a vacuum cold, the cross-sectional area of the inlet port being smaller than the bottom surface area of the porous substrate; (d) the step of bringing the inlet port and the portion of the bottom surface of the porous substrate with plant embryos spread on the corresponding top surface in close proximity to each other when the inlet port communicates with the vacuum source, thereby applying a vacuum to the portion of the bottom surface of the porous substrate immediate proximity of the inlet opening; and (e) moving the inlet opening and the bottom surface of the porous substrate relative to each other below the inlet opening is in communication with the vacuum call until substantially the whole of a desired area of the bottom surface of the porous substrate has been in the immediate vicinity of the inlet opening, each of the inlet openings. from the desired area of the porous substrate on which the plant embryos are spread.
    In one embodiment, the method of the invention further comprises the step of repeating steps (d) and (e) until substantially the entire bottom surface of the porous substrate has been in the immediate vicinity of the inlet port when it is in contact with the vacuum cold.
    In one embodiment, the inlet port is in continuous communication with the vacuum source when the inlet port and the bottom surface of the porous substrate are moved in relation to each other. In one embodiment, the inlet port is in periodic communication with the vacuum cold when the inlet port and the bottom surface of the porous substrate are moved in relation to each other.
    In one embodiment, the inlet port remains stationary, and the porous substrate is moved across the inlet port. In one embodiment, the porous substrate remains stationary, and the inlet port is moved across the bottom surface of the porous substrate.
    In one embodiment, the inlet port is substantially in contact with the bottom surface of the porous substrate.
    In one embodiment, the method of the invention further comprises the step of creating an air flow over and around the embryos scattered on the top surface of the porous substrate when the inlet opening and the bottom surface of the porous membrane are displaced relative to each other when the inlet opening is connected to the vacuum cooling. fluid tan the surface of the scattered embryos via evaporation.
    Porous substrates useful in the practice of the present invention have a pordian ether ranging from about 5 microns to about 1200 microns, from about 50 microns to about 500 microns, from about 70 microns to about 1 micron, from about 100 microns. Am. The porous substrate can have any desired shape and dimension. The shape and dimension of the porous substrate are chosen in order to simplify the handling and suitability for further processing of scattered embryos. Lamp shapes include square, rectangular, or circular shapes. Examples of dimensions are from a surface area of about 25.8 cm2 (4 square inches) to 180.6 cm2 (28 square inches) or more, such as 322.5 cm2 (50 square inches), 645.2 cm2 (100 square inches) up to 3225 , 8 cm2 (500 square inches) or more. Preferred pore substrates are sterilizable and strong enough to resist probe division. Examples of useful porous substrates include membranes, nylon fibers, wire mesh (eg nylon, stainless steel or plastic), natural fibers (eg cotton), paper and polymer fibers. In one embodiment, the porous substrate is a polymeric membrane. In one embodiment, the porous substrate is a nylon membrane.
    To facilitate handling and provision of standing, the porous substrate can be mounted in a frame. The frame can be made of any suitable material such as a lift, such as plastic or metal. In one embodiment, the porous substrate is a nylon membrane and is framed in aluminum.
    The inlet opening, which has a mouth, can be thanked by a housing with the appropriate size and shape of the hoist, such as a rectangular housing with an elongated mouth or a nozzle. In one embodiment, the length of the orifice in the housing is substantially equal to a dimension, such as length or width, of the porous substrate. Typically, the width of the mouth of the housing can range from about 0.00254 cm (0.001 turns) to 2.54 cm (1 turn) or more, as well as from about 0.00254 cm (0.001 turns) to about 0.254 cm (0.1 turns). ), as from about 0.00254 cm (0.001 turn) to about 0.0254 cm (0.01 turn). In one embodiment, the housing has a rectangular structure with an elongated mouth with a length of about 13.3 cm (5.25 turns) and a width of about 0.0051 cm (0.002 turns). Other widths of the mouth of the housing may be suitable, depending on the dimensions of the porous substrate. Plant embryos can be spread on the porous substrate in any arrangement raised and can be distributed to the same extent as raised by the surface area of the porous substrate. Generally, plant embryos can be distributed over a surface of from about 30% to about 90% or more of the surface area of the porous substrate, such as over an area of from about 55% to about 85% of the surface area of the porous substrate.
    A representative example of an automated system useful in practicing the methods of the present invention is shown in Figure 1. Referring to Figure 1, the automated system 10 includes a platform 20 divided into two sections, a porous substrate 30 supported by a surrounding frame, the substrate 30 having a top surface on which embryos are spread and a bottom surface, a mechanical slider arm 40, driven by a motor (not shown) for driving towards the adjacent side of the substrate frame, a vacuum housing 50, which is coated between the two sections of the platform 20, having a narrow elongate orifice 60, a vacuum generator or pump 70 connected to the vacuum housing 50 via a hose 80 and a guide 90.
    In practicing an embodiment of the method of the invention, a porous substrate 30 is placed on a section of the platform 20. Plant embryos may be spread on the porous substrate 30 before it is placed on the platform 20 or after the porous substrate 30 is placed on the platform 20. the mechanical sliding arm 40 projects the porous substrate 30 across the vacuum housing 50. As the porous substrate 30 moves across the vacuum housing 50, the bottom surface of the porous substrate 30 is in contact with the orifice 60 in the vacuum housing 50, while the orifice 60 communicates with the vacuum 70. , which results in liquid being deposited from the porous substrate 30 and air being biased over and around the embryos scattered on the top surface of the porous substrate 30, and through the porous substrate 30.
    Figure 2 schematically shows the porous substrate 30 as it moves from a section of the platform 20 across the orifice 60 to the vacuum housing 50 and 12 to the section on the other side of the platform 20.
    In some embodiments, the negative pressure generated by the vacuum pump used in the practice of the invention may range from about 3.45 kN / m 2 (about -0.5 psi) to about 103.43 kN / m 2 (about -15 psi), as from about 34.48 kN / m2 (about -5 psi) to about 82.74 kN / m2 (about -12 psi). In one embodiment, the negative pressure is about 68.95 kN / m 2 (about -10 psi). In some embodiments, the negative pressure generated by the vacuum source is constant when the inlet port is connected to the vacuum source. In some embodiments, the negative pressure generated by the vacuum source varies when the inlet port is connected to the vacuum source.
    In some embodiments, the porous substrate and the inlet port move relative to each other at a rate in the range of Than about 1 mm / sec to about 45 mm / sec, as from about 1 mm / sec to about 10 mm / sec. In one embodiment, the porous substrate and the inlet port move in relation to each other at a speed of about 3 mm / sec.
    In one embodiment, the vacuum housing 50 with the elongate orifice 60 has a rectangular shape (e.g. rod-shaped) and is sized depending on the size of the porous substrate, so that the vacuum housing 50 with the elongated orifice 60 when used in methods of the invention is in substantially contact with whole of an area of a cross-section of the porous substrate, but will not at contact with the entire surface of the porous substrate at a flag time. In one embodiment, the vacuum housing 50 with the elongate orifice 60 is sized and shaped so that the vacuum housing 50 with the elongate orifice 60 when used in the methods of the invention is in contact with less than 1% of the entire surface of the porous substrate at which time heist, as from about 0.01% to about 0.1`) / 0, as about 0.02% to about 0.05%, as about 0.04%.
    The methods of the present invention focus strongly on a vacuum and a related air flow on the specific surface of the porous substrate which is in the immediate vicinity of, or is substantially in contact with, the mouth of the inlet port when the mouth is in communication with a vacuum cold. Intense focusing of the vacuum on narrow areas or bands on the porous substrate when the inlet port and the porous substrate are moved in relation to each other until substantially the entire porous substrate has been in contact with the inlet port and the vacuum results in a more consistent deposition of liquid over the porous substrate. whole surface. In addition, by means of the methods according to the invention, liquid is removed from a porous substrate on which the growth embryos are spread, without displacing the embryos.
    The methods of the present invention are in contrast to other methods of depositing liquid via a vacuum system. Than porous substrates on which embryos are spread, such as using a Buchner funnel, in that other methods involve simultaneously sucking liquid through the pores throughout the porous area, which can result in uneven fluid deposition over the porous surface and / or displacement of the endbryos. In addition, the present invention enables a faster deposition of liquid than the surface of the porous substrates and the surface of the dispersed embryos compared to previous methods. In known methods, for example, from 1.5 to 7 minutes it was required for sufficient removal of surface liquid on a porous substrate, while the surface liquid can be deposited on less than 1 nnin / porous substrate by using the methods according to the invention. With the current methods, a significant increase in efficiency is obtained, taking into account the requirement for a production set-up for processing hundreds of thousands of embryos.
    In addition, although use of the methods of the present invention may remove liquid from the surface of the dispersed embryos, it is important to note that the methods of the present invention do not substantially affect the moisture content or water potential of the dispersed embryos.
    Once the remaining liquid has been removed from the porous substrate on which the embryos are spread, the embryos may be subjected to further treatment or processing.
    In one embodiment, the plant embryos are pre-cardiac embryos. In one embodiment, the methods of the invention include the steps of (a) culturing embryonic suspension mass in or on a proliferating medium to form pre-cardiac embryos; (b) providing a porous substrate having a top surface and a bottom surface; (c) spreading the pre-cardiac embryos formed in step (a) on the top surface of the porous substrate; (d) providing an inlet port communicating with a vacuum cold, the cross-sectional area of the inlet port being smaller than the bottom surface area of the porous substrate; (e) the step of bringing the inlet port and a portion of the bottom surface of the porous substrate with pre-cardiac embryos spread on the corresponding top surface in close proximity to each other when the inlet port communicates with the vacuum source, thereby applying a vacuum to that portion of the porous surface the substrate located in the immediate vicinity of the inlet opening; and (f) moving the inlet port and the bottom surface of the porous substrate in relation to each other while the inlet port communicates with the vacuum source until substantially the entire desired area of the bottom surface of the porous substrate has been in the immediate vicinity of the inlet port, whereby the liquid is deposited. of the porous substrate on which pre-cardiac embryos are spread.
    In one embodiment, the methods of the invention further comprise the step of transferring pre-cardiac embryos dispersed on the porous substrate, from which liquid has been deposited according to step (f), to a developing medium.
    In one embodiment, the plant embryos are heart-leafed embryos. In one embodiment, the methods of the invention include the steps of (a) culturing pre-cardiac embryos in or on a developing medium to form cardiac embryos; (b) singularizing the heart-leaf embryos produced in step (a); (c) providing a porOst substrate having a top surface and a bottom surface; (d) spreading the singularized heart-leafed embryos at step (b) on the top surface of the porous substrate; (e) providing an inlet port in communication with a vacuum cold, the cross-sectional area of the inlet port being smaller than the bottom surface area of the porous substrate; (f) the step of bringing the inlet port and a portion of the bottom surface of the porous substrate with heart-bladed embryos scattered on the corresponding top surface in close proximity to each other when the inlet port communicates with the vacuum source, thereby applying a vacuum to the portion of the bottom surface of the porous substrate is in the immediate vicinity of the inlet opening; and (g) moving the inlet port and the bottom surface of the porous substrate in relation to each other while the inlet port communicates with the vacuum source until substantially the entire desired area of the bottom surface of the porous substrate has been in the immediate vicinity of the inlet surface of the water opening. of the porous substrate on which heart-leafed embryos are spread.
    In one embodiment, the methods of the invention further comprise the step of subjecting cardiac leaf embryos dispersed on the porous substrate, which liquid has been deposited according to step (g), one or more additional treatments, such as stratification, placement in manufactured seeds and germination.
    The steps in the sonatic embryogenesis process, ie development, stratification and germination, are well known in the field of technology. Examples of media and conditions for each step are described, for example, in U.S. Patent No. 7,785,884. The methods of the invention may be used at any step in the somatic embryogenesis process where it is undesirable to remove supernatant from embryos and / or from a porous substrate. on which embryos are spread.
    Plant embryos which are suitable for use in the methods of the invention may be harrowed from any plant species such as heist, such as dicotyledon or monocotyledonous plants, benign sperms, etc. Conifer embryos are suitable for use in the methods of the invention and may be harrowed without barriers which include in addition, Loblolly pine and Douglas fir.
    Although illustrative embodiments have been shown and described, it will be appreciated that various changes may be made therein without departing from the spirit and scope of the invention.
    权利要求:
    Claims (20)
    [1]
    Providing a porous substrate having a top surface and a bottom surface; 2. spreading growth embryos on the top surface of the porous substrate; Providing an inlet port communicating with a vacuum cold, the cross-sectional area of the inlet port being smaller than the bottom surface area of the porous substrate; (d) the step of bringing the inlet port and a portion of the bottom surface of the porous substrate with plant embryos spread on the corresponding top surface in close proximity to each other when the inlet port communicates with the vacuum source, thereby applying a vacuum to the portion of the bottom surface of the porous substrate immediate proximity of the inlet opening; and (e) moving the inlet port and the bottom surface of the porous substrate relative to each other while the inlet port communicates with the vacuum source until substantially the entire of a desired area of the bottom surface of the porous substrate has been in the immediate vicinity of the inlet port, thereby removing the liquid the area on the porous substrate on which the plant embryos are spread.
    [2]
    A method according to claim 1, wherein the inlet opening is in significant contact with the part of the bottom surface of the porous substrate with growth embryos spread on the corresponding top surface when the inlet opening is in communication with the vacuum cold. 18
    [3]
    The method of claim 1, further comprising repeating steps (d) and (e) until substantially the entire bottom surface of the porous substrate has been inseparable from the inlet port when the inlet port is in communication with the vacuum source.
    [4]
    A method according to claim 3, wherein the inlet opening is continuously in communication with the vacuum cold when the inlet opening and the bottom surface of the porous substrate move in relation to each other.
    [5]
    A method according to claim 3, wherein the inlet port is in periodic communication with the vacuum cold when the inlet port and the bottom surface of the porous substrate move in relation to each other.
    [6]
    The method of claim 1, wherein the inlet port remains stationary and the porous substrate moves across the inlet port.
    [7]
    The method of claim 1, wherein the porous substrate remains stationary and the inlet port moves across the bottom surface of the porous substrate.
    [8]
    The method of claim 1, further comprising the step of creating an air flow over and around the end bridge suns scattered on the top surface of the porous substrate while the inlet port and the bottom surface of the porous membrane move in relation to each other when the inlet port is in contact with the vacuum cold. , which facilitates the removal of fluid from the surface of the dispersed embryos via evaporation.
    [9]
    A method according to claim 1, wherein the inlet opening is filled by a housing with a mouth.
    [10]
    The method of claim 9, wherein the mouth of the housing has a width of from about 0.00254 cm (0.001 inch) to about 0.0254 cm (0.01 turn). 19
    [11]
    The method of claim 1, wherein the inlet port is connected to a vacuum cold with a negative pressure in the range Than of about 3.45 kN / m 2 (about -0.5 psi) to about 103.43 kN / m 2 (about -15 psi). .
    [12]
    A method according to claim 11, wherein the negative pressure in the vacuum cold is constant when the inlet opening is in communication with the vacuum cold.
    [13]
    A method according to claim 11, wherein the negative pressure in the vacuum cold varies when the inlet opening is connected to the vacuum cold.
    [14]
    The method of claim 1, wherein the inlet port and the portisa substrate move in relation to each other at a rate of from about 1 mm / sec to about 45 mm / sec.
    [15]
    The method of claim 1, wherein the porous substrate is a polymeric membrane.
    [16]
    The method of claim 15, wherein the polymeric membrane is a nylon membrane.
    [17]
    The method of claim 1, wherein the pharyngeal embryos are pre-cardiac embryos.
    [18]
    The method of claim 17, further comprising transferring the pre-cardiac embryos to a development medium.
    [19]
    The method of claim 1, wherein the plant embryos are cardiac embryos.
    [20]
    The method of claim 19, further comprising the step of subjecting the heart-leafed embryos to one or more treatments in the form of stratification, insertion into manufactured seeds, and germination. r) 1/2 70 ’- 2/2 1 log
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    Pullman et al.1998|PAC report | March 1998
    同族专利:
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    CA2762823C|2015-05-05|
    CN102559574B|2014-07-30|
    US20120167459A1|2012-07-05|
    US8925245B2|2015-01-06|
    BRPI1106904A2|2013-04-24|
    SE537412C2|2015-04-21|
    CN102559574A|2012-07-11|
    CA2762823A1|2012-06-30|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US5821126A|1986-11-19|1998-10-13|The Regents Of The University Of California|Method for clonal propagation of gymnosperms by somatic polyembryogenesis|
    US4957866A|1989-03-09|1990-09-18|Weyerhaeuser Company|Method for reproducing coniferous plants by somatic embryogenesis|
    US5482857A|1989-03-09|1996-01-09|Weyerhaeuser Company|Method for reproducing douglas-fir by somatic embryogenesis|
    US5041382A|1989-03-09|1991-08-20|Weyerhaeuser Company|High concentration enrichment of conifer embryonal cells|
    US5563061A|1989-03-09|1996-10-08|Weyerhaeuser Company|Method for reproducing conifers by somatic embryogenesis using a maltose enriched maintenance medium|
    US5236841A|1989-03-09|1993-08-17|Gupta Pramod K|Method for reproducing conifers by somatic embryogenesis using stepwise hormone adjustment|
    US5034326A|1989-10-23|1991-07-23|Weyerhaeuser Company|Method for reproducing coniferous plants by somatic embryogenesis using adsorbent materials in the development stage media|
    US5036007A|1989-03-09|1991-07-30|Weyerhaeuser Company|Method for reproducing coniferous plants by somatic embryogenesis using abscisic acid and osmotic potential variation|
    US5294549A|1989-03-09|1994-03-15|Weyerhaeuser Company|Method for reproducing conifers by somatic embryogenesis using mixed growth hormones for embryo culture|
    US5427593A|1990-10-26|1995-06-27|Weyerhaeuser Company|Analogs of botanic seed|
    US6982327B2|1996-05-20|2006-01-03|Cooperatieve Verkoop-En Productievereniging Van Aardeppelmeel En Derivaten Abebe, B.A.|Methods for producing and transforming cassava protoplasts|
    US6548263B1|1997-05-29|2003-04-15|Cellomics, Inc.|Miniaturized cell array methods and apparatus for cell-based screening|
    US6119395A|1997-02-03|2000-09-19|Weyerhaeuser Company|End seals for manufacturing seed|
    AU765726C|1999-08-23|2004-05-20|Weyerhaeuser Company|An embryo delivery system for manufactured seeds|
    US20070099293A1|2005-10-27|2007-05-03|Weyerhaeuser Co.|Use of porous membrane to support developing conifer somatic embryos|
    CN101044839A|2006-03-29|2007-10-03|北京师范大学|Method for breeding seeds with compound ion injected|
    US7785884B2|2006-09-28|2010-08-31|Weyerhaeuser Nr Company|Low density spreading methods for conifer somatic embryogenesis|
    SE534887C2|2007-09-27|2012-02-07|Weyerhaeuser Nr Co|Procedure for stratification and storage of somatic embryos|US9138750B2|2011-12-29|2015-09-22|Weyerhaeuser Nr Company|Spray apparatus and method for separating plant embryos|
    US9688953B2|2014-04-02|2017-06-27|Weyerhaeuser Nr Company|Systems and methods of separating and singulating embryos|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US201061428381P| true| 2010-12-30|2010-12-30|
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