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somatic embryogenesis and embryo harvesting and method and apparatus for preparing plant embryos for plant production

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somatic embryogenesis and embryo harvesting and method and apparatus for preparing plant embryos for plant production


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

Browse recent Arborgen Inc. patents - ,
Inventors: John Joseph CLARK, Narender Singh NEHRA, Mark Russell RUTTER, Jessica S. SAGE, Sydney Keith SEYMOUR, Timothy Joel STOUT, George SURRITTE, Ronald W. WINKLES
USPTO Applicaton #: #20120276634 - Class: 435422 (USPTO) - 11/01/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Plant Cell Or Cell Line, Per Se (e.g., Transgenic, Mutant, Etc.); Composition Thereof; Process Of Propagating, Maintaining, Or Preserving Plant Cell Or Cell Line; Process Of Isolating Or Separating A Plant Cell Or Cell Line; Process Of Regenerating Plant Cells Into Tissue, Plant Part, Or Plant, Per Se, Where No Genotypic Change Occurs; Medium Therefore >Culture, Maintenance, Or Preservation Techniques, Per Se >Involving Conifer Cell Or Tissue (e.g., Pine, Spruce, Fir, Cedar, Etc.)

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The Patent Description & Claims data below is from USPTO Patent Application 20120276634, somatic embryogenesis and embryo harvesting and method and apparatus for preparing plant embryos for plant production.

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This application is a continuation application of U.S. application Ser. No. 12/511,548, filed on Jul. 29, 2009 (U.S. Pat. No. 8,216,841) which is a divisional of U.S. application Ser. No. 11/413,105, filed on Apr. 28, 2006 (U.S. Pat. No. 7,665,243), both of which claim priority to U.S. Provisional Application Ser. No. 60/675,949, filed on Apr. 29, 2005. All applications are incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

Described herein are methods and media for facilitating somatic embryogenesis and for collecting, conditioning, and storing of large numbers of plant embryos prior to germination. Also described herein are a method and apparatus for preparing plant embryos for plant production.

BACKGROUND

Collecting, storing, and conditioning plant embryos, especially somatic embryos, prior to germination are key processes in many aspects of the agriculture industry. The activities necessary for performing these processes, however, are usually performed by hand. For instance, individual embryos are typically transferred to and from various media and vessels and must be plated onto gel media, one by one using forceps and often with the guidance of a dissecting microscope.

Such “hand harvesting” methods are burdensome, time-consuming, costly, and susceptible to contamination. Not only that, but only a limited number of embryos can be collected and treated by a single person during a given period of time. Accordingly, any attempt to increase the number of embryos that can be harvested and subsequently conditioned for germination necessarily requires an increase in manpower, which itself can be costly and often impractical.

An added concern is the inclusion of polyethylene glycol in embryo development media as a osmotic agent. Polyethylene glycol has been incorporated into various media to boost embryogenic development because it is thought to help trigger embryo development. See Fowke et al., Somatic Cell Genetics and Molecular Genetics of Trees, Quebec City, Canada, Aug. 12-16, 1997, which is incorporated herein by reference.

A problem with polyethylene glycol, however, is that it adheres to embryos, possibly interfering with embryo germination. Traditionally, removal of polyethylene glycol is accomplished by storing polyethylene glycol (PEG)-treated embryos on a gel medium without PEG in the cold for a number of weeks. The polyethylene glycol eventually diffuses into the medium away from the embryos. Not surprisingly, this is a time-consuming and burdensome treatment and removal strategy, which imparts an oftentimes unacceptable delay in the overall harvesting and conditioning process.

The agricultural industry and, in particular, the forestry sciences, therefore, are faced with a laborious, expensive, and inefficient method for making, gathering and preparing plant embryos. Such factors prove to be obstacles when operating at commercial levels. And still, hand harvesting is a typically routine practice.

As explained below, however, the present invention provides a robust “Mass Harvesting” method that is rapid and inexpensive. Since Mass Harvesting (MH) minimizes human intervention, it is less susceptible to contamination. Furthermore, the present invention also provides a new way for removing polyethylene glycol. Moreover, the Mass Harvesting method is highly efficient, allowing the simultaneous collection of thousands and hundreds of thousands of plant embryos during a period of time, and can be readily scaled-up for commercial purposes.

In this respect, the present invention also provides a combinatorial approach to exploiting and optimizing genotype-by-treatment interactions of multiple steps in the somatic embryogenesis process.

SUMMARY

In one aspect of the invention, a method for preparing embryos for plant production is provided, which comprises (i) washing multiple plant embryos simultaneously, and (ii) transferring the washed embryos onto a substrate and into an environment suitable for conditioning the embryos for a desired period of time so they become germination-competent for plant production. The method may further comprise retrieving one or more of the embryos at any time point during the desired period of time.

In one embodiment, the plant embryos are somatic embryos. In another embodiment, the embryos are washed on a porous surface. In yet another embodiment, no single embryo has been individually placed by hand onto the porous surface.

In one embodiment, the substrate that is suitable for storing the embryos is a gel, which comprises maltose, glutamine, and abscisic acid. The gel also may contain other ingredients, such as inorganic nutrients. The person of skill in the art of embryo storage and development knows what other ingredients are useful for maintaining and manipulating plant embryos. In another embodiment, the substrate is a filter paper saturated with a volume of liquid media, which comprises maltose, glutamine, and abscisic acid. The gel also may contain other ingredients, such as inorganic nutrients. In another embodiment, the volume of the liquid media that is added to the substrate is 1 ml or 2 ml.

Other conditioning embodiments include, but are not limited to, the following: embryos stored on a gelled medium in cold (1° C. to 12° C., optimally 3 to 6° C.) for varying time (1 day to 24 weeks, optimally from 3 to 12 weeks). During this cold storage the embryos can be placed on a polyester or paper membrane to facilitate subsequent transfer. Embryos on the polyester or paper membrane are then transferred as an entire unit to a vessel and sealed with Nescofilm™, or optionally are placed on top of a dry filter paper within the vessel and sealed with Nescofilm™. Embryos in the sealed vessel are held at room temperature (15 to 30° C., ideally 20 to 28° C.) for varying time (1 to 12 weeks, optimally from 2 to 5 weeks depending on the temperature to which the embryos were exposed during either of the above steps of this conditioning method. That is during: a. cold on a gelled medium and, b. warm in sealed vessel).

In one embodiment, the embryos are stored for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, or more than about 24 weeks.

Another aspect of the present invention is a liquid medium for growing embryonic tissue that comprises a high concentration of casein. A high concentration of casein may be about 900 mg/l, about 1000 mg/l, about 1100 mg/l, about 1200 mg/l, about 1300 mg/l, about 1400 mg/l, about 1500 mg/l, about 1600 mg/l, about 1700 mg/l, about 1800 mg/l, about 1900 mg/l, about 2000 mg/l, about 2100 mg/l, about 2200 mg/l, about 2300 mg/l, about 2400 mg/l, about 2500 mg/l, about 2600 mg/l, about 2700 mg/l, about 2800 mg/l, about 2900 mg/l, about 3000 mg/l, or more than 3000 mg/l. In one embodiment the concentration of casein is between 1100 mg/l and 3000 mg/l.

In one embodiment, the embryonic tissue is from a conifer. In a preferred embodiment, the conifer is pine. In a more preferred embodiment, the pine is Loblolly pine.

In another embodiment, the coniferous tree is selected from the group consisting of Eastern white pine, Western white, Sugar pine, Red pine, Pitch pine, Jack pine, Longleaf pine, Shortleaf pine, Loblolly pine, Slash pine, Virginia pine, Ponderosa pine, Jeffrey pine, Pond pine, and Lodgepole pine, Radiata pine and hybrid crosses thereof. In another preferred embodiment, the coniferous tree is selected from the group consisting of, but not limited to, Abies alba, Abies amabilis, Abies balsamea, Abies bornmuelleriana, Abies concolor, Abies fraseri, Abies grandis, Abies koreana, Abies lasiocarpa, Abies nordmanniana, Abies procera, Araucaria angustifolia, Araucaria araucana, Araucaria bidwillii, Araucaria cunninghamii, Cedrus atlantica, Cedrus deodara, Chamaecyparis lawsoniana, Chamaecyparis pisifera, Cryptomeria japonica, Cuppressocyparis leylandii, Larix decidua, Larix occidentalis, Metasequoia glyptostroboides, Picea abies, Picea engelmannii, Picea glauca, Picea mariana, Picea pungens, Picea rubens, Picea sitchensis, Pinus banksiana, Pinus caribaea, Pinus contorta, Pinus echinata, Pinus edulis, Pinus elliotii, Pinus jeffreyi, Pinus korariensis, Pinus lambertiana, Pinus merkusii, Pinus monticola, Pinus nigra, Pinus palustris, Pinus pinaster, Pinus ponderosa, Pinus rigida, Pinus radiata, Pinus resinosa, Pinus serotina, Pinus strobus, Pinus sylvestris, Pinus taeda, Pinus virginiana, Pseudotsuga menziesii, Sequoia sempervirens, Sequoiadendron giganteum, Taxodium ascends, Taxodium distichum, Taxus baccata, Taxus brevifolia, Taxus cuspidata, Thuja occidentalis, Thuja plicata, Tsuga canadensis, Tsuga heterophylla, and hybrid crosses thereof.

Specific examples of each of such coniferous tree includes: Abies alba, European silver fir; Abies amabilis, Pacific silver fir; Abies balsamea, Balsam fir; Abies bornmuelleriana, Turkish fir; Abies concolor, White fir; Abies fraseri, Fraser fir; Abies grandis, Grand fir; Abies koreana, Korean fir; Abies lasiocarpa, Alpine fir; Abies nordmanniana, Nordman fir; Abies procera, Noble fir; Araucaria angustifolia, Parana pine; Araucaria araucana, Monkeypuzzle tree; Araucaria bidwillii, Bunya pine; Araucaria cunninghamii, Hoop pine; Cedrus atlantica, Atlas cedar; Cedrus deodara, Deodar cedar; Chamaecyparis lawsoniana, Port-Orford-cedar; Chamaecyparis pisifera, Sawara cypress; Cryptomeria japonica, Japanese cedar (Japanese cryptomeria); Cuppressocyparis leylandii, Leyland Cypress; Larix decidua, European larch; Larix occidentalis, Western larch; Metasequoia glyptostroboides, Dawn redwood; Picea abies, Norway spruce; Picea engelmannii, Englemann spruce; Picea glauca, White spruce; Picea mariana, Black spruce; Picea pungens, Colorado blue spruce; Picea rubens, Red spruce; Picea sitchensis, Sitka spruce; Pinus banksiana, Jack pine; Pinus caribaea, Caribbean pine; Pinus contorta, lodgepole pine; Pinus echinata, Shortleaf pine; Pinus edulis, Pinyon pine; Pinus elliotii, Slash pine; Pinus jeffreyi, Jeffrey Pine; Pinus korariensis, Korean pine; Pinus lambertiana, Sugar pine; Pinus merkusii, Sumatran pine; Pinus monticola, Western white pine; Pinus nigra, Austrian pine; Pinus palustris, Longleaf pine; Pinus pinaster, Maritime pine; Pinus ponderosa, Ponderosa pine; Pinus rigida, Pitch pine; Pinus radiata, Radiata pine; Pinus resinosa, Red pine; Pinus serotina, Pond pine; Pinus strobus, Eastern white pine; Pinus sylvestris, Scots (Scotch) pine; Pinus taeda, Loblolly pine; Pinus virginiana, Virginia pine; Pseudotsuga menziesii, Douglas-fir; Sequoia sempervirens, Redwood; Sequoiadendron giganteum, Sierra redwood; Taxodium ascends, Pond cypress; Taxodium distichum, Bald cypress; Taxus baccata, European yew; Taxus brevifolia, Pacific or Western yew; Taxus cuspidata, Japanese yew; Thuja occidentalis, Northern white-cedar; Thuja plicata, Western red cedar; Tsuga canadensis, Eastern hemlock; Tsuga heterophylla, Western hemlock.

In another embodiment, the coniferous plant tissue is a Southern Yellow pine. In yet another embodiment, the Southern Yellow pine is selected from the group consisting of Pinus taeda, Pinus serotina, Pinus palustris, and Pinus elliottii.

The present invention contemplates the Mass Harvesting of somatic embryos from any of these coniferous trees. The present invention is not limited, however, to the Mass Harvesting of only coniferous tree tissues and somatic embryos.

In another embodiment, therefore, the plant tissue, such as embryogenic tissue or a somatic embryo is from a tree selected from the group consisting of chestnut, ash, beech, basswood, birch, black cherry, black walnut/butternut, chinkapin, cottonwood, elm, eucalyptus, hackberry, hickory, holly, locust, magnolia, maple, oak, poplar, red alder, royal paulownia, sassafras, sweetgum, sycamore, tupelo, willow, and yellow-poplar, and intra- and inter-species hybrid crosses thereof. A particularly preferred chestnut for use in the present invention is the American Chestnut.

In one embodiment, the concentration of casein in the liquid medium is about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, or about 3000 mg/l or any integer in between these concentrations.

In one embodiment, the casein is casein hydrolysate.



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stats Patent Info
Application #
US 20120276634 A1
Publish Date
11/01/2012
Document #
13544843
File Date
07/09/2012
USPTO Class
435422
Other USPTO Classes
435431, 435420
International Class
12N5/04
Drawings
17


Embryo
Somatic Embryogenesis


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