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

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

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.

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.

Another aspect of the present invention is a method for obtaining germinating embryos, comprising (i) placing embryogenic cultures from cryostorage onto cryoretrieval medium for a period of time and thereafter growing the embryogenic tissue in liquid medium, (ii) transferring the embryogenic tissue to embryo development medium to generate embryos, (iii) washing a mass of the generated embryos with water, (iv) placing the washed mass of embryos on a substrate that is saturated with conditioning medium, and (v) germinating embryos therefrom, wherein (a) the cryoretrieval medium comprises at least one of a high concentration of casein or an amount of Brassinolide, (b) the liquid medium has a high concentration of casein, (c) the embryo development medium has a desired amount of polyethylene glycol, and (d) the conditioning medium is liquid.

In this method, the liquid medium comprises a concentration of casein which is 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 another embodiment, the percentage of polyethylene glycol in the embryo development medium is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In one embodiment, the percentage of polyethylene glycol in the embryo development medium is 7%. In another embodiment, the percentage of polyethylene glycol in the embryo development medium is 13%.

In one embodiment, the cryoretrieval medium comprises an amount of Brassinolide. In one embodiment, the amount of Brassinolide is 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.10 μM, 0.11 μM, 0.12 μM, 0.13 μM, 0.14 μM, 0.15 μM, 0.16 μM, 0.17 μM, 0.18 μM, 0.19 μM, 0.20, or 0.50 μM. In one embodiment, the concentration of Brassinolide is 0.10 μM.

In another aspect, a method for identifying optimal genotype-specific conditions for embryogenic tissue growth is provided, comprising (i) growing embryogenic tissue that has been retrieved from cryostorage on a medium that comprises an amount of Brassinolide and (ii) comparing the growth of the embryogenic tissue to the growth of embryogenic tissue from the same genotype on media that comprises at least one different amount of Brassinolide.

In another aspect, a method for identifying optimal genotype-specific conditions for embryo production is provided, comprising (i) growing embryogenic cultures on an embryo development medium that comprises an amount of polyethylene glycol and (ii) comparing the growth of the embryogenic cultures into embryos to the growth of embryos from the same genotype on embryo development media that comprises at least one different amount of polyethylene glycol.

In another aspect of the methods disclosed herein are combined to produce a method for identifying optimal genotype-specific conditions for embryogenic tissue growth and embryo production for a particular plant genotype.

In one embodiment, after Mass Harvesting according to any one of these methods, embryos are placed onto a substrate that has been saturated with a volume of liquid conditioning medium, which contains nutrients necessary to prepare the embryos for germination. The substrate may be a filter paper.

In one embodiment, the saturated filter paper onto which the embryos are placed is retained within a dish, such as a Petri dish. In another embodiment, the dish is wrapped with tape or porous wrapping material to control the loss of moisture from the dish. In another embodiment, the dish, which contains the filter paper and the embryos thereon is stored in the cold for a period of time.

The length of time a Mass Harvested somatic embryo can be stored in the cold is from 1 to 5 weeks, for at least 5 weeks, for at least 8 weeks, for at least 10 weeks, for at least 12 weeks, for at least 13 weeks, for at least 14 weeks, for at least 15 weeks, for at least 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, or for more than 24 weeks.

For instance, a Mass Harvested somatic embryo may be stored in the cold under the conditions described herein for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, or 52 weeks, or beyond 52 weeks.

In one aspect of the present invention is a combinatorial method for optimizing somatic embryogenesis, comprising (i) initiating embryogenesis of a plant embryogenic tissue on an initiation medium that comprises a high concentration of casein, (ii) maintaining the initiated embryogenic tissue on a maintenance medium that comprises a high concentration of casein prior to cryostorage, (iii) recovering the embryogenic tissue from cryostorage on a medium that comprises at least one of (a) high concentration of casein or (b) an amount of Brassinolide, and (iv) developing embryos from the recovered embryogenic tissue on an embryo development medium that comprises a percentage of polyethylene glycol that is optimal for the genotype of the embryogenic tissue from which embryos are to developed.

In one embodiment of this method, the percentage of polyethylene glycol in the embryo development medium is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In one embodiment, the percentage of polyethylene glycol in the embryo development medium is 7%. In another embodiment, the percentage of polyethylene glycol in the embryo development medium is 13%.

In another embodiment, the medium onto which the embryogenic tissue is recovered after cryostorage comprises a high concentration of casein and an amount of Brassinolide.

In one embodiment, the amount of Brassinolide is 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.10 μM, 0.11 μM, 0.12 μM, 0.13 μM, 0.14 μM, 0.15 μM, 0.16 μM, 0.17 μM, 0.18 μM, 0.19 μM, or 0.20 μM. In one embodiment, the concentration of Brassinolide is about 0.10 μM.

In another embodiment, the initiation medium further comprises a low concentration of maltose. In one embodiment, the concentration of maltose is about 1 g/l, 2 g/l, 3 g/l, 4 g/l, 5 g/l, 6 g/l, 7 g/l, 8 g/l, 9 g/l, 10 g/l, 11 g/l, 12 g/l, 13 g/l, 14 g/l, 15 g/l, 16 g/l, 17 g/l, 18 g/l, 19 g/l, or 20 g/l. In one embodiment, the concentration of maltose is about 15 g/l.

In another aspect of the present invention is a method for preparing embryos for storage, comprising (i) simultaneously washing multiple plant embryos, and (ii) transferring the washed embryos onto a substrate suitable for conditioning the embryos for storage in a vessel for a desired period of time. In one embodiment, wherein the plant embryos are somatic embryos. In one embodiment, the plant embryos are washed onto a mesh that permits passage of cellular debris and liquid but not the passage of the embryos. Hence, in one embodiment, the embryos are washed on a porous surface and wherein no embryo is placed by hand onto the porous surface. In one embodiment, the step of transferring the washed embryos comprises inverting the mesh on which the embryos were washed directly onto the substrate, wherein the substrate is either already in the vessel or is subsequently moved to a vessel or environment for suitable conditioning and storage. Hence, the embryos may be inverted from the washing mesh and onto a conditioning substrate.

In another embodiment, the conditioning substrate is a gel comprising maltose, glutamine, and abscisic acid. In another embodiment, the conditioning substrate is a filter paper saturated with a volume of liquid media, which comprises maltose, glutamine, and abscisic acid. In one embodiment, the volume of the liquid media is 1 ml or 2 ml.

In one embodiment, conditioning takes place in a high relative humidity environment without cold storage. In another embodiment, conditioning comprises storing the embryos on a gelled medium in the cold for a period of time. In another embodiment, the method further comprises placing the embryos onto a polyester or paper membrane, transferring the membrane to a vessel, which is then sealed, maintaining the vessel at a warm temperature for a period of time.

An aspect of the present invention relates to an apparatus for preparing multiple plant embryos for plant production. The apparatus includes a fluid-delivery structure for delivering input liquid to the multiple plant embryos, a cleaning station in fluid communication with the fluid-delivery structure and configured to hold the multiple plant embryos to receive input liquid from the fluid-delivery structure to clean cellular debris from the multiple plant embryos, an outlet mechanism in fluid communication with the cleaning station and configured to receive output liquid from the cleaning station, and a controller configured to control at least one of the fluid-delivery structure, the cleaning station, and the outlet mechanism.

In an embodiment, the fluid-delivery structure can include a spray mechanism for spraying the multiple plant embryos.

In another embodiment, the cleaning station can include a wash unit for washing the multiple plant embryos, and a rinse unit for rinsing the multiple plant embryos.

In yet another embodiment, the rinse unit can include a porous material configured to hold the multiple plant embryos and having a pore size within a range of 15 microns to 65 microns. The porous material can be configured to hold the multiple plant embryos, the porous material being removable to remove the multiple plant embryos from the rinse unit.

In yet another embodiment, the cleaning station can include a holding unit that transports the multiple plant embryos from the wash unit to the rinse unit. The holding unit can include a porous material in which the pore size can be within the range of 400 microns to 900 microns. The holding unit can include a first porous material configured to hold the multiple plant embryos and having a first pore size. The rinse unit can include a second porous material configured to hold the multiple plant embryos and having a second pore size. Preferably, the second pore size is smaller than the first pore size.

In yet another embodiment, at least one of the fluid delivery structure, wash unit, rinse unit, and holding unit includes a substantially transparent housing to permit monitoring of at least one of washing and rinsing through the substantially transparent housing.

In yet another embodiment, the apparatus includes structure controlled by the controller to move the holding unit from the wash unit to the rinse unit.

In yet another embodiment, the outlet mechanism can include a first outlet in fluid communication with the wash unit and configured to receive output liquid from the wash unit, and a second outlet in fluid communication with the rinse unit and configured to receive output liquid from the rinse unit.

In yet another embodiment, the apparatus can include a negative pressure source in fluid communication with the outlet mechanism to provide a negative pressure. The negative pressure source can include a vacuum system comprising an electronic valve connected to a vacuum pump. The negative pressure source can include a check valve in fluid communication with the cleaning station and configured to operate as a function of output liquid weight and a force of the negative pressure.

In another embodiment, preferably, the controller is configured to control the flow of input liquid through the fluid-delivery structure. The controller can be configured to control the pressure of input liquid delivered by the fluid-delivery structure. The controller can be configured to maintain the impingement of the input liquid within a range of 0.00506 to 0.027 pounds per square inch at a normalized standard distance of twelve inches.

In yet another embodiment, the apparatus can include a negative pressure source in fluid communication with the outlet mechanism, wherein the controller is configured to control a pressure of input liquid delivered by the fluid-delivery structure and to control a pressure supplied by the negative pressure source to the outlet mechanism.

In yet another embodiment, the cleaning station can include a wash unit, and a rinse unit configured to hold the multiple plant embryos. The outlet mechanism can include a first outlet in fluid communication with the wash unit and configured to receive first output liquid from the wash unit, and a second outlet in fluid communication with the rinse unit and configured to receive second output liquid from the rinse unit. The apparatus can further include a negative pressure source in fluid communication with the first and second outlets to supply negative pressure to the first and second outlets, wherein the controller is configured to control the fluid-delivery structure and the negative pressure source.

In yet another embodiment, the apparatus can include a fluid-conditioning system in fluid communication with the fluid-delivery structure and configured to at least one of filter the input liquid and sterilize the input liquid. The fluid-conditioning system can include a membrane filter and a UV sterilizer.

In yet another embodiment, the cleaning station can be configured to remove polyethylene glycol from the multiple plant embryos.

Another aspect of the present invention relates to a method of preparing multiple plant embryos for plant production. The method includes supplying multiple plant embryos in a cleaning station, washing the multiple plant embryos by delivering an input liquid to the plant embryos, and controlling with a controller a flow of input liquid delivered to the plant embryos.

In an embodiment, the impingement of the input liquid can be maintained within a range of 0.00506 to 0.027 pounds per square inch at a normalized standard distance of twelve inches.

In another embodiment, the method can further include supplying a negative pressure to the cleaning station for controlling flow of output liquid, and controlling with the controller the negative pressure supplied to the cleaning station.

In yet another embodiment, the method can further include at least one of filtering the input liquid and sterilizing the input liquid.

In yet another embodiment, the method can include removing polyethylene glycol from the multiple plant embryos in the washing step.

Yet another aspect of the present invention relates to a method of preparing multiple plant embryos for plant production. The method includes supplying multiple plant embryos in a wash unit, washing the multiple plant embryos by delivering a first input liquid into the wash unit, transporting the multiple plant embryos to a rinse unit, rinsing the multiple plant embryos by delivering a second input liquid into the rinse unit, and controlling with a controller at least one of the steps of washing, transporting, and rinsing.

In an embodiment, the method can further include applying a first negative pressure to the wash unit for controlling flow of first output liquid from the wash unit, and applying a second negative pressure to the rinse unit for controlling flow of second output liquid from the rinse unit.

In another embodiment, the method can further include at least one of filtering the first and second input liquids and sterilizing the first and second input liquids.

In yet another embodiment of the method, the first input liquid and the second input liquid can have the same composition. Alternatively, the first input liquid and second input liquid can have different compositions.

Yet another aspect of the present invention relates to a method of preparing multiple conifer somatic embryos for plant production. The method includes positioning the multiple conifer somatic embryos on a porous material having a pore size within a range of 400 microns to 900 microns, and delivering fluid to the multiple conifer somatic embryos on the porous material to clean the conifer somatic embryos. In a further refinement, the pore size of the porous material can be within a range of 560 microns to 710 microns or within a range of 600 microns to 670 microns.

In one embodiment of the present invention at least one of the steps of washing and transferring are automated. Indeed, any one of the methods disclosed herein may comprises steps that are fully or partly automated and/or are computer-operated by software programs that may or may not require human input, intervention, or interaction. In this respect, the present invention also contemplates a fully-automated and semi-automated apparatuses or machines for harvesting embryos. Such an apparatus according to the present invention performs various automated functions pertinent to embryo harvesting techniques of the present invention. Hence, a fully- or semi-automated apparatus of the present invention may perform functions comprising (1) loading of embryos onto a surface, (2) washing of the embryos, (3) rinsing of the embryos, and (4) unloading or transferring of the embryos from the surface to another surface or vessel or container for further manipulation. The apparatus may transfer the treated embryos, by means of a robotic arm or a movable surface, for instance, to a conditioning environment without human intervention. Hence, human intervention may only ever be required at the step of bringing embryos to the apparatus and placing them into or onto the appropriate apparatus surface. From that point onwards, no further human intervention may be necessary until the embryos have been conditioned for a desired period of time. At that point, a human may remove one or more embryos from that conditioning environment to assess whether it is germination competent and then move onwards to plant that germination-ready embryo for plant propagation. Even then, that step, the step of removing the germination-competent embryos can be automated. That is, the apparatus may be designed such that the embryos are automatically removed from the conditioning environment after a period of time that is known to produce germination-competent embryos, and placed onto an appropriate seeding and rooting surface so as to promote germination and shoot growth.

A semi-automated apparatus that performs such functions may be semi-automated in the sense that it may require human intervention at certain points in the process, such as bringing embryos to the apparatus, permitting human intervention to increase or decrease a wash or rinse step, or simply to initiate the computer software that controls the operation of the components of the apparatus. Hence, the present invention contemplates the apparatus that is described herein and which performs the functions outlined above. See also Example 23 below.

The present invention also recognizes and appreciates that certain features of this apparatus can be modified or altered in due course and in response to the embryo harvesting task desired. Hence, the apparatus may be modified so as to increase the total numbers of embryos that can be treated according to the harvesting and washing protocols disclosed herein. For instance, the apparatus disclosed in Example 23, may include more than three units within which to wash embryos. That is, the apparatus may be adapted to include more units or units of larger capacity. Furthermore, the present invention contemplates the manipulation of the computer software that drives and operates the apparatus. In this respect, the present invention contemplates that an automated apparatus of the present invention is controlled by computer software that follows and implements, in computer terms, the process flow diagram depicted in FIG. 11. For instance, the apparatus described herein may be operated by and under the control of computer software that implements the process of FIG. 11. The skilled person appreciates that any of these parameters are open to manipulation. Hence, the present invention contemplates software that controls sensors, which determine the approximate load of embryos that are placed onto a loading surface. Depending on that determination, the software may make and send appropriate computer commands to increase or decrease the length of time of the wash and rinse steps, for example. Hence, if a subsequent batch of embryos is twice that of what was previously loaded, the sensors will direct the duration of the ensuing wash step to be longer or more powerful, or may require the steps of washing and rinsing to be repeated any number of times. Accordingly, the automated apparatus of the present invention for implementing the disclosed and novel harvesting techniques is adaptable, convenient, and useful for simultaneously processing multiple embryos. By multiple embryos, the present invention contemplates that 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 290,000, 300,000 or more, or any integer in between, of embryos can be processed, e.g., washed and rinsed, per day by use of the methods and apparatuses disclosed herein.

The present invention also contemplates embryos that are prepared by any of the methods disclosed herein. In another aspect, the present invention encompasses a plant that is grown from any of the treated embryos disclosed herein.

It is to be understood that both the foregoing general description and the following detailed descriptions are exemplary and explanatory only, and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.

FIG. 1 is a schematic showing the steps from embryogenic initiation, liquid bulk-up, embryo development, Mass Harvesting, cold storage, pre-germination, and germination steps.

FIG. 2 is a schematic drawing of an embodiment of a plant embryo cleaning apparatus according to the present invention.

FIG. 3 is a schematic drawing of a cleaning fluid source, a fluid-conditioning system, and a spray mechanism of the plant embryo cleaning apparatus of FIG. 2.

FIG. 4 is a schematic drawing of a cleaning station of the plant embryo cleaning apparatus of FIG. 2.

FIG. 5 is a schematic drawing of an outlet mechanism and negative pressure source of the plant embryo cleaning apparatus of FIG. 2.

FIG. 6 is a perspective view of the plant embryo cleaning apparatus of FIG. 2.

FIGS. 7A to 7F are perspective views of the of the plant embryo cleaning apparatus of FIG. 2 in operation.

FIGS. 8A and 8B are a cross-sectional view and a side view, respectively, of a spray mechanism, a mounting bracket, and a pneumatic cylinder of the plant embryo cleaning apparatus of FIG. 2.

FIGS. 9A and 9B are a plan view and a cross-sectional view, respectively, of holding units, a mounting bracket, a rotational device, and a pneumatic cylinder of the plant embryo cleaning apparatus of FIG. 2.

FIG. 10A is a plan view of wash units, rinse units, two electronic vacuum valves, and a horizontal moving structure of the plant embryo cleaning apparatus of FIG. 2.

FIG. 10B is a side view of the rinse units, a vacuum manifold, and output funnels of the plant embryo cleaning apparatus of FIG. 2.

FIG. 10C is a cross-sectional view showing the wash units, the output funnels, the vacuum manifold, and a horizontal rail of the plant embryo cleaning apparatus of FIG. 2.

FIG. 10D is a cross-sectional view showing the rinse unit, the output funnels, the vacuum manifold, and the horizontal rail of the plant embryo cleaning apparatus of FIG. 2.

FIG. 11 is a process flow diagram of the intermediate Mass Harvesting machine.

DETAILED DESCRIPTION

The present “Mass Harvesting” method is rapid, inexpensive, and highly efficient. The method entails washing and rinsing large numbers of plant embryos en masse, rather than individually.

After washing, the embryos are transferred to media, which has been formulated herein to increase the integrity and viability of the washed embryos over prolonged periods of time. Furthermore, as described herein, many of the Mass Harvesting and conditioning steps can be performed with liquid media, thereby eliminating certain gel-plating steps and certain storage requirements.

An apparatus is also provided herein to implement the Mass Harvesting method. It is adaptable and can be modified to run automatically, as will be described in further detail below. Briefly, however, the Mass Harvesting apparatus can increase embryo harvesting production rate to a minimum of many tens of thousands of embryos per person/day from about 2000 per day via hand harvesting. This equates to a significant increase in efficiency and an increase the number of germinants and plantable seedlings per gram of starting embryogenic cell cultures. Described below are methods for mass harvesting over 100,000, and even over a million embryos per person per day.

Any collection of embryos can be treated according to the Mass Harvesting method and apparatus. Hence, the present methods do not require a pre-treatment of embryos prior to washing, rinsing, and storing steps. It is useful, however, to appreciate certain pertinent steps and substances that aid the development of embryos. The entire process from cone collection, in the case of conifer treatment, to embryo production, storage, and germination can also be summarized as follows:

1. Cone collection and storage, usually in the cold.

2. Somatic embryogenic initiation on initiation medium.

3. Maintenance of embryogenic tissue on maintenance medium.

4. Cryogenic storage of embryogenic tissue and subsequent cryoretrieval.

5. Growth of embryogenic tissue.

6. Development of somatic embryos on embryo development medium.

7. Harvesting, e.g., via hand or via Mass Harvesting.

8. Conditioning of harvested embryos, may include pre-germination steps.

9. Germination.

Many factors in these culture conditions affect embryo production such as starting material (genotype, source, physiological stage of explant), media (minerals, plant growth regulators, supporting agents), environment (temperature, illumination properties, vessels), timing and finally interaction between all these factors.

In this regard, plant hormones play an important role in embryogenesis. Certain important substances in this respect are auxin and cytokinin.

Abscisic acid (ABA) has long been proposed to play an important role in seed maturation and the suppression of precocious germination. In developing seeds, it stimulates accumulation of reserve substances and prepares embryos for a dormancy. It also increases cold and desiccation tolerance of embryos. In maturing seeds of P. glauca, ABA content is the highest in megagametophytes preceding reserve deposition. Zygotic embryos develop in an environment with high ABA levels, and this hormone might be transported from megagametophytes to embryos. The ABA content varies between 7-30 μM in the embryo and in seed coat cells during seed development.

A decline in sensitivity to exogenous ABA as well as an increase in sensitivity to GAs was observed late in embryo development. Exogenously added ABA inhibits germination, however, during seed development embryos are able to germinate despite the high ABA levels. Partial drying will increase germination and decrease the ABA level. Additional drying continues to accelerate germination, but does not decrease ABA concentration further. Water availability may affect sensitivity. These changes in hormone sensitivity may play a role in germination.

ABA also has an important role during somatic embryogenesis. In somatic embryos of Picea glauca, for instance, ABA stimulates embryo growth and inhibit precocious germination, and in somatic embryos of P. glauca×P. engelmannii, ABA treatment enhances storage protein accumulation. Exogenous ABA is also capable of inducing the expression of genes coding some LEA proteins in somatic embryos of Picea glauca and Pinus edulis. Sometimes, without ABA treatment, abnormal fast-growing somatic embryos may develop. This type of somatic embryo is usually ungerminable because of inadequate preparation for germination.

Some spontaneous development of somatic conifer embryos may exist in a hormone-free medium or with PEG treatment, but in an ABA-containing medium, midway in embryo development the embryos begin to accumulate triglycerides and storage proteins and develop to mature cotyledonary somatic embryos. The first non-spontaneous maturation of coniferous somatic embryos in of P. abies using a low level of exogenous ABA (0.1-1 μM) was reported by Becwar M. R., et al., “A method for quantification of the level of somatic embryogenesis among Norway spruce callus lines,” Plant Cell Reports, 6: 35-38, 1987, which is incorporated herein by reference. See also U.S. Pat. No. 5,183,757, which is incorporated herein by reference.

Higher levels of ABA were later used in embryo maturation of P. abies and P. sitchensis and up to 100 μM ABA levels are used in conifer embryo cultures. See, for instance, von Arnold S. & Hakman I., “Regulation of somatic embryo development in Picea abies by abscisic acid (ABA),” J. Plant Physiol., 132: 164-169, 1988, Boulay et al., “Development of somatic embryos from cell suspension cultures of Norway spruce (Picea abies Karst.),” Plant Cell Rep., 7: 134-137, 1988, and Attree S. M. & Fowke L. C., “Embryogeny of gymnosperms: advances in synthetic seed technology of conifers,” Plant Cell Tiss. Org. Cult., 35: 1-35, 1993.

ABA can be used in standard initiation medium. A concentration of 10 mg/l of ABA is not atypical. See, for instance, U.S. Pat. No. 5,677,185, which is incorporated herein by reference.

Gibberellins also have an important role in embryogenesis. More than 12 GAs have been identified in conifers (Wang et al. 1996). Exogenously added GAs do not have any apparent influence on development of somatic embryos probably due to sufficient synthesis of endogenous GAs.

According to the present invention, a high concentration of casein, which is a well known source of nitrogen, also is beneficial in initiation media, maintenance media, and liquid “bulk-up” media. Other sources of nitrogen may also be beneficial in such media such as glutamine.

In somatic or asexual embryogenesis, somatic cells may develop into plantlets following the same morphological steps as zygotes. In vitro somatic embryos are induced either directly from the explant or indirectly through the subculturable callus or suspension culture stage.

The first success in somatic embryogenesis among conifers was reported in 1985 for Picea abies (Norway spruce). See Hakman I. & von Arnold S., “Plantlet regeneration through somatic embryogenesis in Picea abies (Norway spruce),” J. Plant Physiol., 121: 149-158., 1985, and Chalupa V., “Somatic embryogenesis and plantlet regeneration from cultured immature and mature embryos of Picea abies (L.) Karst.,” Comm. Inst. Forest Chech., 14: 57-63, 1985.

Somatic embryogenesis for Pinus is described in Gupta P. K. & Durzan D. J., “Somatic polyembryogenesis from callus of mature sugar pine embryos,” Bio/Technol., 4: 643-645, 1986.

Similar treatments have enabled somatic embryogenesis in several other conifer species. See Minocha S. C. & Minocha R., “Historical aspects of somatic embryogenesis in woody plants,” in SOMATIC EMBRYOGENESIS IN WOODY PLANTS, Vol 1: 9-22, Kluwer Academic Publishers, The Netherlands. ISBN 0-7923-3035-8, 1995.

In vitro proliferation of conifer embryogenic cultures usually takes place on auxin and cytokinin containing culture media. Organic nitrogen, sometimes in the form of casein, is also often needed to maintain embryogenic capacity of cultures. Events in early development of a conifer somatic embryo are currently being heatedly debated. Observations range from initiation of embryo development from long, vacuolated or small, dense cytoplasmic cells via unequal division to embryonic and suspensor initials.

For effective embryo production, embryogenic tissue cultures of conifers are usually maintained on an auxin and cytokinin-containing medium, unlike many dicotyledonous embryogenic cultures, where only the auxin (usually 2,4-D) is often needed to induce embryogenesis.

For further discussion on embryogenesis, see Chapter 2 of Santanen, A., “Polyamine Metabolism During Development of Somatic and Zygotic Embryos of Picea Abies (Norway Spruce),” Academic Dissertation, University of Helsinki, Faculty of Science, Department of Biosciences, Division of Plant Physiology, November 2000.

Accordingly, it is well know how to appropriately stimulate embryogenic cultures and embryo production from a variety of plant species, and the substances that are useful for enhancing or facilitating these biological developments.

In this regard, it has been discovered herein that Mass Harvest washing and rinsing substantially removes polyethylene glycol molecules that adhere to embryo surfaces during their exposure to embryogenic development media. This is a significant discovery because the removal of polyethylene glycol via washing and rinsing eliminates several time-consuming and burdensome steps in the traditional harvesting protocol. For example, it is not necessary to store Mass Harvested embryos on gelled medium in the cold for 3-4 weeks to allow diffusion of polyethylene glycol away from the embryos.

In certain situations prior to Mass Harvesting, it is desirable to “bulk-up” embryogenic tissue before transferring onto an embryo development media. Traditionally, embryogenic tissue cultures that have been cryogenically-stored, for instance, are plated onto gelled medium and incubated for a period of time until there is sufficient growth to justify their transfer to a development medium. For instance, embryogenic tissue can be grown on polyester rafts placed on the surface of gelled medium. The tissue, plus the raft, can be frequently transferred to fresh medium, e.g., every two weeks, until a suitable tissue mass has been achieved. Cultures can typically be incubated in the dark at 25° C. The methods can be used from growing tissue derived from immature seed explants, or from tissue retrieved from cryopreservation. Suitable media are described in Table 1 and 2.

Cryostorage of cultures can use media of the standard method (using DCR liquid medium) or the alternative method formulated herein using Mi3 liquid medium with high casein and, optionally, high glutamine. The use of the Mi3 medium with increased casein and high glutamine results in significant increased growth on cultures over the standard methods. The cryostorage media also may contain two supplements sorbitol 0.4 M, and DMSO (Dimethyl Sulfoxide) 10% by volume. See, for instance, U.S. Pat. No. 5,413,930.

One may also include ABA (10 mg/liter) in the cryorecovery medium. See U.S. Pat. No. 6,682,931.

According to the present invention, however, the embryogenic tissue may be “bulked-up” or grown in a liquid version of the traditional gel medium. Consequently, eliminating the plating step helps to streamline the embryo development process and reduce costs associated with making the gel plates, for instance. In this alternative method, liquid suspension cultures are established by initially dispersing embryogenic tissue in liquid media in an appropriately sized flask or culture vessel.

Suspension cultures are incubated in the dark at 25° C. on a shaker table. Additional liquid suspension medium can be routinely added during the incubation period. Cultures can be monitored weekly until they have grown to a mass that is suitable for plating for embryo development. In this regard, the “settled cell volume” (SCV) is an indicator of liquid-suspended cell mass. In this case, when the SCV reaches at least 50% of the total suspension volume, the embryogenic tissue is at a suitable mass for plating. If additional tissue is needed, suspension cultures from single flasks can be used to establish additional flasks. Suitable media are described in Tables 1 and 2.

Embryogenic tissues that have been bulked up from either the traditional gel or the alternative liquid suspension media can be used to develop somatic embryos. An amount of the bulked up tissue can be transferred to a polyester raft and placed onto the surface of embryo development medium. The tissue and rafts can be transferred to fresh medium after a period of time. Typically, the bulked-up tissue can be stored on embryo development media for 4-6 weeks in the cold. After that time, the embryos can be Mass Harvested according to the protocols described herein.

Embryo production for individual cell lines can vary depending on the particular embryo development used. Therefore, it is possible to appropriately optimize the embryo development media for developing embryos from certain species and/or to increase the proportion of cell lines that produce embryos. In this regard, described herein are different percentages of polyethylene glycol that have been found to enhance the development of embryos from different conifer genotypes.

While these particular bulk-up and embryo development steps exemplify how embryogenic tissue can be treated prior to Mass Harvesting, the present Mass Harvesting method can be used to process any collection of embryos regardless of their prior condition of development and storage.

The Mass Harvesting procedure and apparatus may entail placing embryos onto a sieve, filter, or some other kind of mesh. The species and condition of the embryos can be taken into consideration when choosing which mesh size to use in order to capture appropriately-staged embryos. Pine somatic embryo dimensions are generally of length about 1.0 mm to about 5.0 mm and the diameter ranged from about 0.5 mm to about 2.0 mm. Accordingly, the person of skill in the art would know what would be suitable mesh sizes to use in order to manipulate embryos but prevent losing an unsuitable number of embryos by virtue of their falling through too-large openings in the mesh. Typical commercial mesh sizes have a grid with openings ranging from 500 to 1000 microns. Smaller sizes also can be used, such as those with pore sizes of 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 670, 700, 710, 750, and 800 microns or any integer in between. In certain cases, 800 microns is too large for certain conifer cell lines. Since high polyethylene glycol concentrations yield smaller embryos, it may therefore be desirable to use sieve size(s) that have pores smaller than 670 microns.

Sterile water is sprayed onto or poured onto the embryos on sieve to wash away embryo development media and embryogenic cellular debris. This wash step may be repeated any number of times. The washed embryos can then be transferred to media suitable for storage or germination.

Contrast this method with the traditional hand harvesting method, which entails manually picking individual embryos and placing them onto a gel medium plate, which is then stored in the cold for 3-4 weeks. In this regard, the “standard” method typically used to store and germinate harvested embryos can be described as follows:

Standard method: The standard method comprised two main steps, A and B, they are:

Step A (i) somatic embryos are harvested from embryo development plates and placed onto gelled medium for a period of time of cold storage, where the plates housing the embryos are sealed to prevent or reduce moisture loss from the plates, (ii) the embryo plates are then placed in a high relative humidity environment for a period of time, and; Step B subsequently transferred to gel embryo germination medium and singulation of somatic embryo onto fresh germination plates. Germinated embryos are then transferred to a vessel for conversion.

As evidenced from the data related in the following Examples, the present Mass Harvesting method makes certain steps of this standard method, such as the placement in a high relative humidity environment, unnecessary.

Washing and rinsing a mass of embryos can be facilitated by connecting various vessels to a water source and to a vacuum pump, which can draw a continuous and even flow of sterile water over the embryos. The Vacuum Manifold, Nalgene Product no. DS0345-0001 is an example of one type of vacuum system useful for the present invention.

Accordingly, one may devise an “embryo-washing unit,” which comprises holes or at least one porous membrane or side or surface onto which embryos can be placed and into which water can flow through. For instance, the embryo-washing unit can be a cylinder within which is located a sieve or mesh or some kind of filter onto which the embryogenic tissue is placed. See the “Harvesting unit” in described in connection with Example 23.

The unit may be connected to a vacuum pump. In this regard, the unit may be connected to another device, such as a funnel, which in turn is appropriately connected to a manifold port typically used to draw a vacuum and/or fluid from a water source.

In this case, with the vacuum on, one may direct a spray nozzle at the embryos housed in the embryo-washing unit and begin washing the embryos. Debris and other materials will be drawn through the sieve or filter and the porous surface or hole of the unit and directed into a drain or container along with the waste water. The cellular debris may be sent directly to a drain instead of collecting it on a polyester trap placed within the vacuum manifold.

Once it is apparent that washing has removed most of the debris associated with the mass of embryos and the embryos have been thoroughly washed, the embryos can be removed to a clean filter paper and rinsed with sterile water.

Mass Harvesting resulted in a 12-fold increase in embryo harvesting efficiency over Hand Harvesting. One person can now readily harvest 30-embryo development plates per hour or 240-plates per day. Assuming that each plate contains 100 somatic embryos, it will amount to 24,000 embryos harvested by one person per day using Mass Harvesting procedure compared to 2000 embryos handled by one person per day with Hand Harvesting procedure.

Further, any number of wash and rinse units can be operationally linked together or connected to a vacuum manifold and water supply. In one example, the Mass Harvesting apparatus has 3 wash units and 3 collection units that operate simultaneously (see Example 23). The wash water is provided by the cold tap water system passing through an electric solenoid valve and a UV sterilizer. The solenoid valve is controlled by an adjustable timer which is activated with a foot pedal by the operator. Waste water is handled by a vacuum assisted drain line connected to house vacuum though a water/air separator (Vac-Stack). Vac-Stack collects water and drains to the building drain system without user intervention. Vacuum is used in both the wash step and the collection step.

Such an arrangement is useful for efficient Mass Harvesting of embryos for small scale clonal work and mid scale production 100K-500K plants. It follows that different magnitudes of embryo and plant production can be achieved depending on the number of discreet arrangements and apparatuses and numbers of shifts per 24-hour day are employed in any given period of time. The system is compatible with current development and conditioning methods. Efficiency of this system is over 100,000 embryos per person per day with a line that produces about 100 embryos per plate.

The apparatus also can be readily adapted for automation. Hence, one or more systems can Mass Harvest millions of embryos per day per person and automatically prepare these embryos for conditioning. The system consists of a conveyer belt on which the tissue and embryo slurry is delivered gradually using a pump. The embryos are then separated from the tissue using the sterile water spray. The washed embryos are air dried using a vacuum and dislodged into a conditioning unit.

In any of these arrangements, the embryo-washing unit can be inverted and the embryos transferred to a pre-existing polyester raft on another unit that is connected to the same vacuum manifold. This can be accomplished manually or via single-function (e.g., hard) or multi-function (e.g., programmable or flexible) automation. The embryos on the polyester raft can then be rinsed with the sterile water.

Once satisfactorily rinsed, the embryo-loaded polyester raft can then be transferred to a labeled plate of storage or conditioning media. One particularly useful conditioning medium is 2M21. See Tables 1 and 2 for the composition of 2M21.

Typically, conditioning medium is a gel encased within a dish, such as a Petri dish. The present inventive method however employs a new liquid-based version of the medium, whereby embryos are placed onto a filter paper that has been saturated with liquid medium. The embryos may be placed directly onto the saturated filter paper. Alternatively, the embryos can be placed onto a membrane, for instance, which is then placed onto the saturated filter paper, where the membrane is permeable in some respect to the liquid or to the moisture in the filter paper. In distinction to those typical conditioning procedures, therefore, the present inventive nutrient-rich, liquid-based approach neither desiccates nor starves the embryos.

To prevent loss of moisture from the Petri dish-plated form of embryos, the dish may be sealed with any one of a number of tapes or wrappings. For instance, dishes may be sealed with Nescofilm™ when harvesting is done. These plates then can be stored for a desired period of time in the cold, i.e., at 4° C., although storage under these conditions is not always necessary. For instance, it may be desirable to bypass an entire cold storage step and proceed straight to germination. It is not necessary to “starve” the embryos during cold storage or at any other point in this process.

This generic procedure of mass washing, rinsing, and transferring the embryos to storage or conditioning media can be repeated for each collection of embryos, although care should be exercised to ensure that new embryo-washing units and vacuum connections are replaced with each new cell line.

Returning to the nine-step general process for taking a conifer cone through somatic embryogenesis and germination described above, Table 1 provides the pertinent media that can be used at each particular step.

In the standard method for initiation and maintenance of embryogenic tissue, “WV5” and gelled and liquid “DCR” media are used. Embryo development medium is denoted by “MSG.” The conditioning, pre-germination, and germination steps are conducted on media designated as “2M21” and “MODMS.” Accordingly, the generic process can be rewritten thus:

1. Cone collection and storage, usually in the cold.

2. Somatic embryogenic initiation on initiation medium (WV5 gel).

3. Maintenance of embryogenic tissue on maintenance medium (DCR gel).

4. Cryogenic storage of embryogenic tissue (in DCR liquid) and retrieval thereof.

5. Growth of embryogenic tissue (on DCR gel).

6. Development of somatic embryos on embryo development medium (on MSG).

7. Harvesting, e.g., via hand or via Mass Harvesting.

8. Conditioning of harvested embryos (on 2M21 gel).

9. Germination (on MODMS gel).

An improved method, which is disclosed herein, includes a “liquid bulk-up” medium and other casein-rich media, “Mi3,” which enhance embryogenesis. Accordingly, the new nine-step method can be rewritten thus:

1. Cone collection and storage, usually in the cold.

2. Somatic embryogenic initiation on initiation medium (WV5 gel with high casein concentration).

3. Maintenance of embryogenic tissue on maintenance medium (Mi3 gel with high casein concentration).

4. Cryogenic storage of embryogenic tissue (in Mi3 liquid with high casein concentration optionally with a genotype-specific amount of Brassinolide).

5. Growth of embryogenic tissue on cryoretrieval medium (on Mi3 gel with high casein concentration).

6. Liquid bulk-up to enhance growth of embryogenic tissue (in liquid Mi3).

7. Development of somatic embryos on embryo development medium (on MSG gel optionally with genotype-specific polyethylene glycol concentrations).

8. Harvesting, e.g., via hand or via Mass Harvesting.

9. Conditioning of harvested embryos (on a substrate saturated with 2M21 liquid).

10. Germination (on MODMS gel).

The Mi3 medium contains a base level of 500 mg/l of casein, but the total amount of casein in the Mi3 medium may be 0.5, 1.0, 1.5, 2.0, 2.5, 2.6, 2.7, 2.8, 2.9, or up to 3.0 grams per liter.

Secondly, the MSG medium, which is used for embryo development may contain varying levels of polyethylene glycol. It has been found herein that different levels of polyethylene glycol affect different Pine genotypes differently. Accordingly, it may be necessary to optimize the level of polyethylene glycol in the MSG medium to match the growth development characteristics of the genotype in question.

In this regard, the Mi3 medium also may contain an optimal amount of Brassinolide. Brassinolide, which was first isolated from rapeseed plant pollen (Brassica napus L.), is a naturally occurring plant steroid that promotes growth, increases yields for grain and fruit crops, and makes plants more resistant to drought and cold weather. Related compounds, called brassinosteroids, are found in a wide variety of plants, and also can be used to augment the embryo development medium.

Thirdly, the conditioning of harvested embryos is useful for preparing the embryos for germination and prolonged storage is conducted on a substrate, such as a filter paper, that is saturated with 2M21 liquid. One such method involves cold conditioning and slow moisture loss from a moist filter paper substrate during the exposure to cold. It was effective from 8 to 16 weeks. The highest germination and conversion rates were obtained when embryos were conditioned in cold for 8 weeks on moist filter paper with 1 ml of liquid 2M21 medium.

According to the present invention, all pre-germination steps and embryo germination steps can be conducted via such a medium in a single dish without touching the embryos. Thus the embryos will be touched only once when they are transferred to a sterile or non-sterile vessel in order to induce photoautotrophic conversion into planting stocks. Conversion is not limited to a particular type of vessel. Indeed, the embryos may be placed into any environment that induces their photoautotrophic conversion into suitable planting stocks. Reducing the extent of manual interaction with embryos in such fashion will significantly reduce media and labor costs for embryo germination process.

Steps 8 and 9 of the preceding modified process can also be elaborated upon as follows:

Mass Harvesting conditioning: (A) mass harvested mature somatic embryos are placed onto filter paper that has been saturated with an appropriate volume of 2M21 liquid media to facilitate saturation and placed in cold storage for a desired number of weeks, with a mechanism in place for controlling moisture loss from the filter paper over time, and (B) transferring the embryos to gel embryo germination medium and singulation of somatic embryo onto fresh germination plates. Germinated embryos are then transferred to a vessel for conversion.

Other embodiments include (1) high casein concentration in initiation, maintenance, bulk-up, and cryoretrieval media; (2) Brassinolide in cryoretrieval medium; (3) storing Mass Harvested embryos in high relative humidity without a cold storage step; (4) storing Mass Harvested embryos on filter paper saturated with liquid media in the cold, with various methods for altering the rate of moisture removal from petri dish/moistened filter paper; (5) the development of genotype-specific embryo development medium with optimal percentages of polyethylene glycol and/or Brassinolide.

Examples of the media that can be used according to the present invention are described in detail in Table 1, but their more pertinent ingredients can be summarized as follows. All of the media described herein contain inorganic salts and vitamins as detailed in Table 2. Where “casein hydrolysate” is used it is at a desired high concentration. That is, the concentration of casein hydrolysate that is detailed in Table 1 is 500 mg/l, but this is the base amount. More casein hydrolysate is typically added, e.g., an additional 0.5 to 2.5 mg/l, to the base media to provide the high concentration. The choice of how much extra casein to add is dependent on the condition of the embryos and the genotype being treated, which can be deduced empirically.

Initiation medium (gel): myo-inositol, casein hydrolysate, maltose, 2,4-D, BAP, ABA.

Maintenance medium (gel): myo-inositol, casein hydrolysate, sucrose, 2,4-D, BAP, glutamine, glycine.

Maintenance medium (liquid): myo-inositol, casein hydrolysate, sucrose, 2,4-D, BAP, glycine, activated carbon.

Bulk-up medium (gel and liquid): myo-inositol, casein hydrolysate, sucrose, 2,4-D, BAP, ABA, glutamine, glycine, activated carbon.

Embryo development medium (gel): maltose, ABA, glutamine, polyethylene glycol, activated carbon.

Conditioning medium (gel and liquid): maltose, ABA, glutamine.

Germination medium (gel): sucrose, activated carbon.

Of course, any suitable media and method for conditioning and germinating Mass Harvested embryos can be used, not only those media and methods described herein. Similarly, and as previously noted, any method of obtaining embryos, particularly somatic embryos, can be used to provide embryos for treatment according to the Mass Harvesting methodology and with the apparatus described herein.

In the southern U.S. approximately 1 billion seedlings of southern pine are produced per year. These seedlings are currently derived from seed orchard seedlings utilizing 50 years of tree improvement. Even with the increased genetic gain from this traditional tree improvement approach, the Forest Products and Paper Industry need higher yielding trees with improved wood properties. To meet industry needs requires clonal loblolly pine be implemented on large scale. Somatic Embryogenesis (SE) is the one large-scale propagation technology capable of both capturing the genetic gain from traditional tree improvement, and meeting large-scale clonal production needs of Forest Products and Paper Industry.

Although improvements have been made in conifer Somatic Embryogenesis (SE), no comprehensive approach has been formulated or developed to an operational scale that ensures the efficient capture of genotypes from conifer species. In particular, this limitation or lack of efficient genotype capture impacts implementation of SE with the Pinus species that have proven recalcitrant to efficient clonal propagation by SE.

The present invention therefore provides a combinatorial approach that enables one to take advantage of large genotype by treatment interactions in a sequential step-wise manner. The result of this approach is an optimized protocol for large-scale production that is customized for each genotype.

The conifer somatic embryogenesis is a multi-step process as described above and as illustrated in FIG. 1. The steps may be classified according to the following sequential order: culture initiation, culture maintenance or establishment, cryogenic storage, cryo-retrieval, multiplication or tissue bulk up, embryo production, embryo harvesting and conditioning, embryo germination and conversion to planting stock.

Optimization of particular parameters of any given step can improve the efficiency of that particular step in the regeneration process. And it is known that genotype by treatment interactions exist for specific steps in the somatic embryogenesis process noted above.

The present invention provides the sequential application of a combinatorial approach to exploiting the genotype by treatment interactions of multiple steps in the somatic embryogenesis process. The results reported here show that one can make very significant increases in capture and efficiency by using a combinatorial approach screening genotypes to improving the SE process in conifers.

Following is a summary of experiments described in the Examples.

1. It was demonstrated that for mass harvested somatic embryos the gelled 2M21 medium used for cold treatment and PEG block removal can be replaced with 2 ml of liquid 2M21 medium.

2. Pre germination conditioning of somatic embryos in the presence of liquid 2 M21 medium at room temperature or in cold could serve as a substitute for current high relative humidity treatment

3. The use of 3M-filter tape allowed pre-germination conditioning of somatic embryos by avoiding moisture condensation in Petri plates at both room temperature and in cold.

4. Cold treated somatic embryos conditioned in the presence of liquid 2M21 medium can be effectively induced to begin the germination process by addition of 2 ml liquid MODMS1 germination medium

5. Cold storage of embryos on a new liquid 2M21 medium showed potential to extend embryo storage to at least 24 weeks while retaining good embryo quality, whereas storage of embryos on gelled 2M21 medium per the standard method decreased embryo quality after 16 weeks or longer

6. The properly conditioned germination ready somatic embryos are amenable to extended cold storage by double wrapping with 3M-filter tape and Saran wrap.

7. An experiment comparing different embryo conditioning methods showed that a “new” cold conditioning method provides a reasonable alternative to the standard cold plus high relative humidity conditioning protocol.

8 Storing harvested embryos on gelled medium during the cold conditioning phase followed by holding embryos in sealed vessel at warmer temperatures worked as effectively as a standard method that necessitates holding embryos over water in vessels during the warm phase. The new alternative method is simpler and more amenable to large-scale conditioning for commercial production.

9. Germination and conversion were similar or slightly higher for several new cold conditioning treatments compared to the standard method.

10. Addition of 1 ml liquid 2M21 medium added to filter paper may be better than 2 ml for the 8 week duration of cold conditioning.

11. The cold conditioning method noted here results in slow moisture loss during the extended (8 to 16 weeks) depending on the volume of water added to the filter paper. This allows for flexibility in scheduling when to go to germination.

12. A liquid medium for growing embryogenic tissue that comprises a high concentration of casein.

13. An improved method for retrieving embryogenic tissue from cryostrorage by including either or both high casein and brassinolide in the tissue recovery medium and tissue bulk up medium.

14. Using a battery of media at several sequential steps, results in a combinatorial approach to increase the likelihood of maximizing the number of commercial candidate cell lines for scale-up, and also increasing efficiency and reducing cost for implementing the somatic embryogenesis process.

All of the media that are referenced below, e.g., “WV5,” “DCR,” “Mi3,” “MSG,” “2M21,” and “modMS” are detailed in Tables 1 and 2.

Procedure

Following is one strategy for collating, washing, and rinsing embryos according to the “mass harvesting” concept presently described.

Place the embryo-washing unit on top funnel on washing side (washing and rinsing sides are determined by preference) of the manifold port.

Working with a single line at a time, load embryos into the washing unit using a spatula.

Turn on vacuum port to washing side of manifold.

Position spray nozzle over embryo-washing unit and depress foot pedal control to begin washing.

Wash embryos until all suspensor tissue is separated from the embryos (embryos will remain on mesh surface of the embryo-washing unit while the tissue is washed into drain bottle. Release foot pedal and turn off vacuum.

Place a polyester raft in the funnel on the rinsing side of the manifold port.

Once washing is complete and excess water has drained, invert embryo-washing unit and transfer to the rinsing side of the manifold.

Position spray nozzle over embryo-washing unit and depress foot pedal control to begin rinsing embryos onto polyester raft.

Remove polyester raft from washing side of manifold and replace.

Transfer the embryo-washing unit back to washing side of the vacuum manifold.

Lift the embryo loaded polyester raft and transfer to a labeled plate of 2M21 media.

Wrap the plates with Nescofilm™ when harvesting is done.

Repeat this procedure for the remaining plates.

Change funnel tops and embryo-washing unit with each new cell line.

Media

See Tables 1 and 2 for detailed recipes for various media used in the present invention.

Materials

2.95 inch circular polyester raft supports with 35 micron pores (SEFAR 07-33/10)

Steri 350™ heat sterilization device

Dumont SS non-magnetic pointed forceps, 5.5″ long, 6 inch dissecting forceps and spatula

Nescofilm™ strips or 6″×5″ perforated Saran plastic wrap

Polypropylene Buchner funnels—90 mm I.D. tops (cut down to ½ inch height) with 71 mm long stems (fitted with #8 black rubber stoppers)

Nalgene 3 port stainless steel manifold that holds 3 Buchner funnel stems simultaneously (center port is not used) and has individual stopcock controls for applying a vacuum (equipped with the appropriate tubing for connecting to waste Carboy and the vacuum supply)