1. Field of the Invention
This invention relates to methods for propagating plants. More particularly, it relates to methods for handling, sowing, and germinating plant somatic embryos.
2. Description of Related Art
Considerable attention has been given to the development of somatic embryogenesis processes for clonal reproduction of plants and consequently, the specific steps of somatic embryogenesis have been documented in the art for a wide diversity of plant species including both gymnosperms and angiosperms. All methods of somatic embryogenesis are known as tissue culture processes and generally commence with the selection of an explant from a desired plant. The explant is removed from the parent plant tissue by excision and is subsequently cultured on at least one medium to produce a cell mass capable of further differentiation and development. The cell mass can be maintained and proliferated in the undifferentiated state indefinitely, or manipulated to stimulate differentiation into immature somatic embryo structures which can then be cultured further into mature embryos (see, for example, U.S. Pat. Nos. 4,957,866; 5,238,835; 5,294,549; 5,491,090; 5,501,972; 5,563,061; 5,677,185, as well as PCT Publication No. WO 96/37096, all of which are hereby incorporated by reference). Matured somatic embryos can be harvested and germinated immediately, or dried and then germinated, or dried and stored until required for germination (for example, refer to U.S. Pat. Nos. 5,183,835; 5,238,835; 5,413,930; 5,464,769, as well as PCT Publication No. WO 96/37095, all of which are hereby incorporated by reference).
Tissue culture media used to proliferate and propagate plant cultures through the various stages of somatic embryogenesis are typically enriched with mixtures of nutrients that are specifically formulated for each plant species and for the various stages of somatic embryogenesis. A common problem encountered with all somatic embryogenesis processes is microbial, i.e., bacterial, fungal, yeast, contamination of the media and/or plant explants and/or the resulting embryogenic cultures. Microbial contaminants compete with the embryogenic cultures for the nutrients in the media, and in many cases, will infect, consume, parasitize, or otherwise pathogenize the cultures. Consequently, steps must be taken to prevent microbial contamination from the beginning of the embryogenesis process when the tissue explants are excised from the parent tissues, through production, harvesting, drying and germination of the somatic embryos and their subsequent growth into fully functional transplants, i.e., somatic seedlings which can be transplanted into soil or horticultural growing mixes. All manipulations of the cultures at each step of the somatic embryogenesis processes are typically done using aseptic techniques. Embryogenic cultures which show any evidence of microbial contamination at any step in somatic embryogenesis process are sterilized and discarded.
Two of the greatest barriers to commercializing somatic embryogenesis technologies are the processes of sowing and germinating plant somatic embryos. Although numerous protocols are known for the sowing and germination of somatic embryos and growing them into intact functional seedlings, none of these protocols have demonstrated compatibility with conventional horticultural equipment and practices.
Generally, the known protocols for germinating somatic embryos fall into two categories. The first is sowing naked, i.e., uncoated, somatic embryos using aseptic techniques, onto sterilized semi-solid or liquid media contained within a solid-support to facilitate germination (e.g., U.S. Pat. Nos. 5,183,757; 5,294,549; 5,413,930; 5,464,769; 5,506,136) and subsequently, transplanting the germinants into conventional growing systems. The most significant disadvantage of such protocols for sowing naked somatic embryos is that each embryo typically must be handled and manipulated by hand for the germination and transplanting steps. Although various automation options including robotics and machine vision, have been assessed for their usefulness in cost-effective reduction or elimination of the extensive hand-handling currently necessary to sow naked embryos (Roberts et al., 1995), no commercial equipment currently exists which can reliably, aseptically, and cost-effectively perform the in vitro protocols for germination of naked somatic embryos and subsequent transplanting into conventional propagation systems.
The second category of protocols teach encapsulation of somatic embryos (e.g., U.S. Pat. Nos. 4,777,762; 4,957,866; 5,183,757; 5,482,857) to provide a means by which the embryos can presumably be sown with mechanical devices such as seeders and fluidized drills, into conventional growing systems. However, there are a number of disadvantages with gel-encapsulated somatic embryos. For example, the hydrated semi-solid physical characteristics of encapsulated embryos make them incompatible for use with conventional seeding equipment currently available for commercial plant propagation, because the semi-solid gel-encapsulated somatic embryos tend to clump together during handling and consequently, are difficult to singulate and dispense. Furthermore, compositions of encapsulated embryos prepared as taught by the art, clog-up the conventional equipment, and for these reasons, it currently is not possible to sow encapsulated embryos with conventional seeding equipment. Consequently, novel equipment has been developed specifically for delivery of encapsulated somatic embryos into conventional growing systems. Such sowing devices have been reviewed by Sakamoto et al. (1995), but these devices have only been developed and tested as prototypes. Because of mechanical limitations and the high costs associated with the prototype mechanical seeders developed for sowing encapsulated embryos, none are currently available for commercial acquisition and use.
Another disadvantage with encapsulated somatic embryos is the lack of nutrient availability that is characteristically supplied to zygotic embryos by their attendant endosperm or megagametophyte tissues. Consequently, the encapsulation technology for somatic embryos has been extended to include the incorporation of various nutrients such as sugars, fertilizers, oxygen, into the encapsulation medium (e.g., Carlson and Hartle, 1995; U.S. Pat. Nos. 4,583,320; 5,010,685; 5,236,469, all of which are herein incorporated by reference). However, a distinct disadvantage associated with nutrient-amended encapsulated embryos is their susceptibility to microbial invasion during manufacture, storage, and during germination if germinated on non-sterile media.
Furthermore, it must be pointed out that although considerable prior art (e.g., PCT Patent Application WO 94/24847, and U.S. Pat. Nos. 5,010,685; 5,236,469; 5,427,593; 5,427,593; 5,451,241; 5,486,218) teaches methods to manufacture xe2x80x9cartificial seedsxe2x80x9d consisting of somatic embryos encapsulated in gels, which may or may not be amended with nutrients, and which may or may not be encased within a rigid covering, and although the prior art makes references to sowing said artificial seeds ex vitro into germination media comprised of soil or soilless mixes, the prior art only teaches methods for germinating said artificial seeds in vitro, i.e., on sterilized semi-solid laboratory media. No methods are taught or otherwise disclosed, in the prior art for sowing said encapsulated somatic embryos and/or manufactured and/or artificial seed into conventional growing systems using conventional sowing equipment.
However, the most significant disadvantage with all prior art taught for encapsulating or otherwise coating somatic embryos, is that somatic embryos processed following those protocols typically have, as a consequence, much lower germination vigor and success than corresponding zygotic seeds (Carlson and Hartle, 1995). Carlson and Hartle (1995) concluded that considerable research is still required before xe2x80x9cmanufacturedxe2x80x9d or xe2x80x9cartificialxe2x80x9d seeds based on encapsulation and/or coating of somatic embryos will have practical utility. However, it should be noted that the germination vigor of naked, i.e., uncoated or non-encapsulated somatic embryos produced with methods disclosed in the art can approximate those of the corresponding zygotic seeds (e.g., greater than 85%) (Gupta and Grob, 1995).
An object of the present invention is to facilitate the production of seedlings from somatic plant embryos.
Another object of the invention is to produce pre-germinated somatic embryos of plants that can subsequently or immediately be planted and grown into seedlings.
The present invention relates to a multi-step process to produce seedlings from somatic embryos which begins by germinating somatic embryos and then placing the resultant germinants into dormancy for extended periods of time (e.g. at least 24 hours), for example by drying and/or cooling the embryos, or merely storing them without contact with nutrient solutions. This first component of the multi-step process is referred to as xe2x80x9cpre-germination.xe2x80x9d It has surprisingly been found that pre-germinated embryos placed into a state of physiological dormancy, can be sown and re-germinated when desired or convenient, ex vitro using conventional seeding equipment, into a wide variety of horticultural nursery containers filled with various types of non-sterile growing mixes commonly used in commercial horticultural and agricultural plant propagation.
In one form of the invention, there is provided a process of producing a somatic seedling from a somatic embryo, said process comprising the steps of: pre-germinating a somatic embryo by placing the somatic embryo in contact with a liquid medium used for germinating somatic embryos to produce a pre-germinated somatic embryo, optionally partially immersing the pre-germinated somatic embryo in a solution of abscisic acid (ABA), optionally drying the pre-germinated somatic embryos, placing the pre-germinated somatic embryo on or within the surface of a three-phase substrate, said phases comprising solid, liquid and gas phases, placing said substrate containing said pre-germinated somatic embryo into an environmentally-controlled plant-growing environment, controlling at least one environmental factor in said environment during germination of the pre-germinated somatic embryo to facilitate re-germination, growth and development of the pre-germinated somatic embryo, and applying water and/or nutrient solutions at regular intervals during said period of somatic embryo re-germination to the surface of the substrate in the form of microdroplets such that pre-germinated somatic embryo re-germination, growth and development occur.
Preferably, the somatic embryo is placed in contact with said liquid medium for a period of time in the range of 2-30 days, the medium contains sucrose in a range of 1-9%, the pre-germinated somatic embryo is immersed in said ABA solution for a period of time in the range of xc2xd-2 hours, and the pre-germinated somatic embryo is dried to a moisture content in the range of 5-75%.
The invention, in another aspect, includes a process of producing a pre-germinated somatic embryo, which comprises: pre-germinating a somatic embryo by placing the somatic embryo in contact with a liquid medium used for germinating somatic embryos, optionally partially immersing the pre-germinated somatic embryo in an ABA solution, and optionally drying the pre-germinated somatic embryo. The pre-germinated somatic embryos are preferably placed in a state of physiological dormancy.
The invention also includes a process of producing plant seedlings, which comprises sowing pre-germinated somatic embryos produced by the above process in a three-phase substrate, and growing said pre-germinated somatic embryos. Water and nutrients are preferably applied to a surface of said three-phase substrate in the form of microdroplets, at least to the stage at which the embryos become autotrophic. At this stage, the volume of water or nutrients may be reduced or eliminated altogether.
The invention includes pre-germinated somatic embryos and grown seedlings produced by the above processes.
The process of germinating and then harvesting germinated somatic embryos for re-sowing, is referred to as xe2x80x9cpre-germination.xe2x80x9d
It has also surprisingly been discovered that pre-germinated somatic embryos placed into physiological dormancy, can be desiccated to moisture contents in the range of 5-76%. Furthermore, it has been discovered that desiccated pre-germinated somatic embryos can be stored for extended periods of time without significant declines in physiological integrity or re-germination potential. It has also been discovered that desiccated pre-germinated somatic embryos are amenable for sowing with conventional seeding equipment into conventional plant propagation media for re-germination and further growth and development using conventional plant propagation practices.
Consequently, the preferred multi-step process of the present invention includes, but is not limited to, the steps of pre-germinating somatic embryos, harvesting the pre-germinated embryos, placing the pre-germinated somatic embryos into a state of physiological dormancy, sowing the pre-germinated physiologically dormant somatic embryos onto or into germination media, propagating the sown pre-germinated somatic embryos in environmental conditions manipulated to facilitated imbibition, germination, and development into complete seedlings possessing shoots and roots. Furthermore, the multi-step process may also include, if so desired, a step during which pre-germinated somatic embryos are (a) re-sown immediately after harvesting, or (b) desiccated prior to re-sowing.
An advantage of the present invention, at least in preferred forms, is that it may provide a process by which a somatic embryo can be germinated, harvested from the germination medium, and subsequently sown and re-germinated ex vitro using conventional horticultural and agricultural equipment, containers, growing substrates, and growing environments. Alternatively, after the germinated somatic embryos are harvested, they can be dried and stored for periods of time prior to sowing and re-germination.
Another advantage of the invention, at least in preferred forms, is that it may provide a process by which the germination of somatic embryos followed by harvesting and subsequent ex vitro sowing and re-germination of the germinants, can be practiced with a diverse variety of gymnosperm and angiosperm species. Alternatively, harvested pre-germinated somatic embryos of both gymnosperm and angiosperm species may be desiccated and stored for periods of time prior to ex vitro sowing and re-germination.
There are several additional advantages inherent with the use of the process of the invention, at least in its preferred forms. For example, one advantage of pre-germinating plant somatic embryos is that they generally show exceptional vigor during re-germination and subsequent development into complete seedlings possessing shoots and roots. Furthermore, desiccated pre-germinated somatic embryos are particularly useful for preserving the physiological viability of the embryos during extended storage prior to sowing and re-germination. Yet another advantage of pre-germinating somatic embryos is that they can be sorted according to size, length and shape to facilitate production of more uniform crops after sowing, re-germination and growth.
A key advantage of the present process, at least in preferred forms, is that all components of the multi-step process can be practiced in conventional plant propagation environments without the need for aseptic handling processes for sterile growing environments. More specifically, aseptic procedures, and sterile or sanitized equipment and germination/growing environments are not required for successful germination, desiccation, storage, sowing and re-germination of somatic embryos and their subsequent development into complete functional seedlings, thus enabling the entire pre-germination, sowing and re-germination steps to be performed, if so desired, in commercial plant propagation or greenhouse or nursery growing facilities. However, it is preferable to perform the first step, i.e., germination of somatic embryos, in sterile in vitro conditions.
Another advantage is that the pre-germinated somatic embryos can be sown with conventional seeding equipment such as but not restricted to, vacuum-drum seeders, fluid-drill seeders or needle-jet seeders.
A further advantage is that commonly used horticultural and agricultural products such as, but not restricted to, soil-less seedling mixes or rock wool or foams, can be used as the supports onto which the pre-germinated somatic embryos are sown and subsequently re-germinate into and penetrate with their roots.
Yet a further advantage is that if necessitated by the conditions in the commercial growing environments, existing commercial pesticide products such as, but not restricted to fungicides, bactericides, antibiotics, nematicides, insecticides and the like, which are registered for use with the plant species from which the somatic embryos are produced, can be applied to the sown pre-germinated somatic embryos per label instructions for effective pest control, or alternatively, applied to the growing substrates prior to sowing the somatic embryos.
Another advantage is that exogenous nutrients necessary for successful somatic embryo germination and re-germination can be applied via the various numerous methods, such as misting, fogging, spraying, watering and drenching. Furthermore, the exogenous nutrients can be applied in conjunction with conventional horticultural fertigation practices.
A number of terms are known to have differing meanings when used in literature describing this art. The following definitions are believed to be ones most generally used in the fields of botany, plant somatic embryogenesis, and are consistent with the usage of the terms in the present specification.
xe2x80x9cABAxe2x80x9d is abscisic acid, a plant growth regulator.
An xe2x80x9cexplantxe2x80x9d is the organ, tissue or cells derived from a plant and cultured in vitro for the purposes of starting a plant cell or tissue culture.
An xe2x80x9cembryogenic culturexe2x80x9d is a plant cell or tissue culture capable of forming somatic embryos and regenerating plants via somatic embryogenesis.
xe2x80x9cSomatic embryogenesisxe2x80x9d is the process of initiation and development of embryos in vitro from somatic cells and tissues.
A xe2x80x9csomatic embryoxe2x80x9d is an embryo formed in vitro from vegetative (somatic) cells by mitotic division of cells. Early stage somatic embryos are morphologically similar to immature zygotic embryos; a region of embryonal cells subtended by elongated suspensor cells. The embryonal cells develop into the mature somatic embryo.
A xe2x80x9czygotic embryoxe2x80x9d is an embryo derived from the sexual fusion of gametic cells.
xe2x80x9cMegagametophytexe2x80x9d is haploid nutritive tissue of gymnosperm seed, of maternal origin, within which the gymnosperm zygotic embryos develop.
xe2x80x9cEndospermxe2x80x9d is haploid nutritive tissue of angiosperm seed, of maternal origin, within which the angiosperm zygotic embryos develop.
A xe2x80x9cclonexe2x80x9d when used in the context of plant propagation refers to a collection of individuals having the same genetic constitution, and are produced from a culture that arises from an individual explant.
A xe2x80x9clinexe2x80x9d is another term for xe2x80x9cclonexe2x80x9d.
xe2x80x9cNutrientsxe2x80x9d are the inorganic micro- and macro-minerals, vitamins, hormones, organic supplements, and carbohydrates necessary for culture growth and somatic embryo germination.
A xe2x80x9cmicrodropletxe2x80x9d is a self-contained unit of liquid (e.g. water or water-based solution) that is smaller than a drop of the same liquid allowed to form by gravity from a nozzle or solid surface, and is generally contained within a collection of similar microdroplets (e.g. a cloud, mist, fog, fine spray, or the like) produced by applying pressure (e.g. air, a gas or a liquid flowing under pressure provided by a pump) to a drop or other body (e.g. a stream) of the liquid. A microdroplet is usually less than half the size (diameter), and may be less than a quarter or tenth of the size, of a drop of the same liquid, and is preferably small enough to remain temporarily suspended in air (i.e. as an aerosol), and to drift with air currents, rather than fall directly to the ground.
xe2x80x9cAutotrophicxe2x80x9d refers to the stage of plant development when the photosynthetic organelles and related enzymes and biochemical pathways are fully functional and capable of converting light energy, atmospheric carbon dioxide and water into the pre-requisite carbohydrates (e.g., glucose) necessary to sustain further plant growth and development.
xe2x80x9cPhysiological dormancyxe2x80x9d refers to the cessation of the normal metabolic processes, i.e., anabolism and catabolism, that are inherent in plant growth and development, in a manner that does not negatively affect viability.
xe2x80x9cImbibitionxe2x80x9d is the absorption and/or adsorption of water by certain colloids present in seeds or embryos, which results in the swelling of the tissues and activation of enzymatic and physiological processes.
xe2x80x9cGerminationxe2x80x9d is a process of development leading to the emergence of a radical or epicotyl or hypocotyl or a root from an embryo and filter development into a complete seedling having shoots and root.
xe2x80x9cPre-germinationxe2x80x9d is the partial germination of somatic embryos which are harvested and subsequently sown into non-sterile growing media for ex-vitro re-germination, or alternatively, desiccated and stored prior to sowing and re-germination.
In a preferred form, the present invention is generally a multi-step process for ex vitro sowing and germination of plant somatic embryos using conventional horticultural equipment and facilities, comprising, but not restricted to, some or all of the following sequential steps:
1. Sowing the plant somatic embryos onto physical supports placed within containers which contain a liquid germination medium, said embryos placed onto the physical supports in a manner such that the embryos are not submerged in the liquid medium, but instead, such that the liquid medium forms a thin capillary layer around the embryo, said capillary layer also referred to as a film.
2. Incubating the somatic embryos surrounded with a film of germination medium for a period of time ranging between 2-30 days such that embryo germination commences as evidenced by the emergence of a shoot and a root.
3. Placing the containers with the somatic germinants into cold storage, said cold storage comprised of temperatures in range of 2-15xc2x0 C., preferably in the range of 4-10xc2x0 C., for at least, but not limited to, 1 day.
4. Conditioning the somatic germinants by transferring them from the containers containing germination medium to containers containing an ABA solution in the range of 2-100 xcexcM, preferably in the range of 5-20 xcexcM, for a period of time ranging between 30-180 minutes, preferably in the range of 60-90 minutes.
5. Drying the conditioned germinants by transferring them from the containers containing the ABA solution, to a drying chamber, said drying chamber maintaining a relative atmospheric humidity in the range of 35-99% RH, preferably in the range of 80-99%, and incubating said conditioned germinants in said drying chamber for a period of time ranging between 12 hours to 7 days, preferably in the range of 18 hrs to 48 hrs.
6. Storing said conditioned and dried embryos in a sealed package in a facility wherein the temperature is maintained in the range of xe2x88x9285xc2x0 to 30xc2x0 C., preferably in the range of 2-20xc2x0 C.
7. Sowing the pre-germinated plant somatic embryos into nursery containers containing a three-phase substrate, said three phases comprising solids, liquids and air.
8. Placing the nursery containers sown with plant somatic embryos, into a conventional plant propagation environment in which light, temperature, atmospheric humidity, and moisture content of the rooting substrate can be controlled and manipulated to enable and facilitate the re-germination of the somatic embryos and their further development into seedlings.
9. Supplying an aerosol to the surface of the nursery containers sown with somatic embryos, said aerosol containing the necessary carbohydrate compounds required to initiate and sustain the re-germination processes of the somatic embryos.
10. Supplying in the forms of an aerosol and/or a liquid suspension and/or a liquid solution, the micro- and macro-mineral elements required to sustain the re-germination of somatic embryos and their subsequent development into seedlings.
11. Adjusting as required during the somatic embryo re-germination period, the ambient light intensity and diurnal photoperiod, temperature and atmospheric humidity to maintain the development of re-germinated somatic embryos into fully functional seedlings.
Alternatively, at the completion of step 2, pre-germinated embryos may be sown directly after harvesting, i.e., per steps 7-11.
A particular advantage of the process, at least in its preferred forms, is that special hygienic and/or aseptic and/or sterile handling methods and/or equipment and/or facilities are not required to successfully handle, sow and germinate, dry, and re-germinate plant somatic embryos. Accordingly, these steps may be carried out in non-sterile, unhygienic and/or septic conditions, preferably using xe2x80x9cnakedxe2x80x9d embryos (i.e., non-encapsulated or otherwise coated embryos).
It is preferable that the invention be practiced with plant somatic embryos that have been dried to moisture contents that approximate those of their corresponding zygotic seeds, i.e., in the range of 5-20% and more specifically, in the range of 10-15%. However, it is possible to practice the present invention with somatic embryos containing higher moisture contents in the range of 20-70% with the only limitation on the upper limit being the highest level of moisture content that the somatic embryos can be singulated with conventional seeding or seed-handling equipment.
It is preferable that the pre-germination step is carried out in a container wherein there is a physical support such as, but not restricted to, filter paper or a screen comprised of a nylon or polypropylene material or other such materials, is placed on the liquid germination medium such that plant somatic embryos are held on the surface or above the surface of the liquid medium such that a thin capillary layer or film of the germination medium is formed around the somatic embryos. Alternatively, the somatic embryos can be successfully pre-germinated on discontinuous physical substrates comprised of materials such as but not limited to, vermiculite, perlite, peat, coconut husk fibres and the like, said discontinuous supports containing sufficient liquid germination medium to enable the formation of a thin capillary layer or film of germination medium around the somatic embryos. The somatic embryos can be sown onto the surface of the absorbent material by hand or by the means of a mechanical sowing device such as but not restricted to conventional seeding equipment.
However, it is also possible to accomplish the pre-germination of somatic embryos by sowing them with conventional seeding equipment into empty multi-chambered nursery containers exemplified by but not restricted to miniplug trays, said containers having their drainage holes covered by a mesh-like material which will support the somatic embryos after sowing. The containers are then placed onto liquid germination media such that the somatic embryos are in contact with but are not submerged in the liquid media, such that a thin layer of film of germination medium is formed around the somatic embryos.
Although the pre-germinated and subsequently dried somatic germinants can be sown with all conventional seeding equipment used for sowing zygotic seeds, it is preferred to use equipment that dispenses singulated seed into multi-chambered nursery containers, commonly referred to as miniplug trays or cell-packs, said containers commonly used to produce plant plugs which can be mechanically transplanted into larger containers or into field-growing environments.
Another important advantage of the present invention, at least in its preferred forms, is that the sowing and propagation of pre-germinated somatic embryos can be practiced with a wide variety of non-sterilized growing substrates commonly used in conventional plant propagation. The preferred growing substrate is peat-based and has been formulated specifically for germination of zygotic seed and is exemplified by mixtures such as (a) 15.2 cu.ft of peat, 8 cu.ft. of vermiculite, 680 grams of dolomite lime, and 300 grams of Micromax(copyright) (a commercial fertilizer composition comprised of microelements such as but not limited to sulfur, boron, manganese, magnesium, cobalt and iron), and (b) 16.2 cu.ft. of peat, 6.75 cu.ft. perlite, 4 cu.ft. vermiculite, 6 kilograms of dolomite lime, 1.5 kilograms of gypsum, 375 grams of potassium phosphate, 250 grams Micromax(copyright), and 35 grams of wetting agent. Alternatively, commercially formulated mixes such as PRO-MIX-G(copyright) or PRO-MIX-PGX(copyright) (Premier Peat Moss Ltd. Montreal, PQ, Canadaxe2x80x94these are commercial soilless plant growing media comprised of mixtures containing but not limited to peat, perlite, vermiculite and pumice), Sunshine Mix #3 (Sun-Gro Horticulture Inc., Hubbard, Oreg., USA), and Redi-Earth(copyright) (The Scotts Co., Marysville, Ohio, USAxe2x80x94this is a commercial soilless plant growing media comprised of mixtures containing but not limited to peat, perlite, vermiculite and pumice) can also be used with the present invention. It is preferred that the peat-based growing substrate is moistened to a moisture content in the range of 59-75% and then dispensed into multi-chambered trays commonly used for production of plant plugs. Although examples of such trays include Styrofoam #252 miniplug trays manufactured by Beaver Plastics Inc (Edmonton, AB, Canada) and hard plastic #288 or #512 miniplug trays manufactured by TLC Polyform Inc (Plymouth Minn., USA, 55441), the present invention can be practiced with other such multi-chambered trays, or alternatively, with individual pots. It should be noted that the practice of the present invention is not restricted to peat-based mixtures, but also includes other substrate such as Jiffy-7 peat plugs, composted or shredded coconut husk fibres commonly referred to as xe2x80x9ccorxe2x80x9d or xe2x80x9ccoirxe2x80x9d (1993 Crystal Co., St. Louis, Mo., USA), polymerized substrates (Grow Tech Inc., San Juan Bautista, Calif. USA; Preforma Inc., Oberlin, Ohio USA), extruded foams such as Oasis( (Smithers-Oasis Ltd., Kent, Ohio, USAxe2x80x94this is a commercial expanded foam product comprised of urea formaldehyde), rock wool (Rockwool International A/S, Hovedgaden 584, DK-2640, Denmark) and the like. Regardless of the rooting substrate chosen, it""s physical characteristics should enable development and maintenance of a high relative humidity in the gaseous phase, i.e., in excess of 75% RH, within the substrate while minimizing saturation of the substrate with the liquid phase.
After the pre-germinated somatic embryos are sown onto the surfaces of the rooting substrates, if desired, the embryos may be covered with a thin layer of additional rooting substrate that may be comprised of the same material underneath the embryos or alternatively, with a different type of material. One non-limiting example is sowing the pre-germinated embryos onto PRO-MIX-PGX medium, then overlaying the embryos with a thin layer of coconut husk fibres.
Nursery containers sown with pre-germinated somatic embryos are preferentially placed into a conventional plant propagation environment wherein the conditions are within but not limited to the ranges of temperatures of 15-35xc2x0 C., relative humidities of 75-100%, light intensities of 10-500 foot candles, and diurnal cycles of 6 h day/18 h night-22 h day/2 h night.
It is preferable to maintain a very high level of atmospheric humidity around the nursery containers sown with pre-germinated somatic embryos, i.e., greater than 90% RH, for the first 3-7 days after sowing to facilitate somatic embryo imbibition and germination. A number of methods can be used to maintain the atmospheric humidity at these levels including but not restricted to placing the containers in a greenhouse environment with misting or fogging equipment which is deployed at controlled intervals, placing the containers in a fogging or misting tent or chamber, placing clear plastic domes over the nursery containers and then removing domes periodically to mist or fog the sown embryos and replacing the domes immediately thereafter. Another non-limiting method is to provide a space ranging between 2 mm and 10 mm above the surface of the rooting substrate onto which the embryos are sown and the top of the container, and then covering the top of the nursery container with a plastic film which is removed to enable misting or fogging of the sown embryos and then immediately replaced. After somatic embryo germination is established as evidenced by development of epicotyl and root structures, the germinants can be weaned from the high relative humidity environments and integrated into conventional nursery cultural practices by gradually reducing the amount of misting/fogging applied and/or by extending the periods of time between the misting or fogging steps.
It is preferable to maintain the sown pre-germinated embryos in a high relative humidity environment, i.e., greater than 90% RH, for a period of, but not restricted to, 3-7 days after sowing to facilitate embryo imbibition, prior to supplying exogenous nutrients required for embryo germination.
Another important feature of the invention, at least in preferred forms, is that the exogenous nutrients, including but not restricted to carbohydrates and minerals, required for successful somatic embryo re-germination and subsequent growth and development may be applied as aerosols. The nutrient solutions may be applied with, but not restricted to, conventional misting and/or fogging equipment. Although, the nutrients can be applied individually or combined into one solution, it is preferred to supply the carbohydrates as one solution and the remaining nutrients as a separate solution. A non-limiting example of how this can be practiced is by applying a 3% sucrose solution as a mist to the surface of the growing substrate containing a sown pre-germinated embryo, and then applying at a later time, a solution containing a mixture of mineral nutrients formulated to deliver 454 mg/l nitrogen, 81 mg/l phosphorus, 704 mg/l potassium, 50 mg/l calcium, 39 mg/l magnesium, 193 mg/l sulfur, 3 mg/l manganese, 0.5 mg/l zinc, 89 mg/l chlorine, 3 mg/l iron, 0.7 mg/l iodine, 0.6 mg/l boron, 0.01 mg/l molybdenum, 0.01 mg/l cobalt, and 0.01 mg/l copper. Alternatively, the macronutrients can be supplied as a commercial formulation such as but not restricted to PlantProd(copyright) Plant Starter Fertilizer 10-52-10 (nitrogen-phosphate-potassium) or PlantProd(copyright) Forestry Seedling Starter 11-41-8 (nitrogen-phosphate-potassium) (Plant Products Ltd., Brampton, ON, Canadaxe2x80x94these are commercial water-soluble fertilizers containing mineral nutrients such as nitrogen, phosphorus and potassium, and a dye).
An alternative non-limited means of supplying exogenous nutrients to pre-germinated somatic embryos sown onto three-phase growing media within nursery containers is to irrigate or xe2x80x9cdrenchxe2x80x9d the media with nutrient solutions formulated as previously described. This is preferably done just before the embryos are sown in the three-phase growing media.
Since microorganisms such as fungi, bacteria, yeast, and algae, are ubiquitous in conventional plant propagation substrates, equipment, containers and growing environments, a wide variety of chemical and biological pesticide products are available to control and eradicate plant pathogens. It has surprisingly been found that aseptic handling procedures and sterilized growing substrates, nursery containers and environments are not required to successfully germinate and grow plant somatic embryos. Indeed, the invention can be practiced in conventional plant propagation environments using only the standard commercial methods of hygiene. Furthermore, we have surprisingly found that pesticides such as Benlate(copyright) (a commercial fungicide composition containing a chemical active ingredient), Rovril(copyright) (a commercial fungicide composition containing a chemical active ingredient), Trumpet(copyright) (a commercial insecticide composition containing a chemical active ingredient) and the like, which are registered for pest control in plant crops, can be used in conjunction with somatic embryos pre-germinated and subsequently sown with the present novel multi-step procedure.
The following Examples are provided to further illustrate the present invention and are not to be construed as limiting the invention in any manner.