Source: http://www.google.com/patents/US5486218?ie=ISO-8859-1
Timestamp: 2014-12-25 01:39:51
Document Index: 580458854

Matched Legal Cases: ['Application No. 0', 'in fine', 'Application No. 0', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2']

Patent US5486218 - Oxygenated analogs of botanic seed - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsAn analog of botanic seed is disclosed which comprises a plant embryo preferably encapsulated, or at least in contact with, a hydrated oxygenated gel. The gel can be oxygenated by passing oxygen gas through a gel solution before curing the gel or by exposing the gel to oxygen gas after curing. The gel...http://www.google.com/patents/US5486218?utm_source=gb-gplus-sharePatent US5486218 - Oxygenated analogs of botanic seedAdvanced Patent SearchPublication numberUS5486218 APublication typeGrantApplication numberUS 08/069,560Publication dateJan 23, 1996Filing dateJun 1, 1993Priority dateOct 26, 1990Fee statusPaidAlso published asUS5236469, US5451241Publication number069560, 08069560, US 5486218 A, US 5486218A, US-A-5486218, US5486218 A, US5486218AInventorsWilliam C. Carlson, Jeffrey E. Hartle, Barbara K. BowerOriginal AssigneeWeyerhaeuser CompanyExport CitationBiBTeX, EndNote, RefManPatent Citations (34), Non-Patent Citations (86), Referenced by (9), Classifications (11), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetOxygenated analogs of botanic seedUS 5486218 AAbstract An analog of botanic seed is disclosed which comprises a plant embryo preferably encapsulated, or at least in contact with, a hydrated oxygenated gel. The gel can be oxygenated by passing oxygen gas through a gel solution before curing the gel or by exposing the gel to oxygen gas after curing. The gel is preferably oxygenated by adding to an uncured gel solution a suitably stabilized emulsion of a perfluorocarbon compound or a silicone oil, which compounds are capable of absorbing large amounts of oxygen, and are non-toxic and inert. An analog of botanic seed can further comprise an outer shell at least partially surrounding the gel and embryo, thereby forming a capsule. The outer shell preferably is shaped to aid the radicle of a germinating embryo in protrusively rupturing the capsule, thereby facilitating successful germination and minimizing incidence of seedling malformation. Other shell materials are selected to provide requisite rigidity to the capsule while imparting minimal restriction to successful germination.
We claim: 1. An analog of a botanic seed comprising totipotent plant tissue encapsulated in a substantially non-phytotoxic hydrated gel, the hydrated gel comprising a perfluorocarbon compound so as to enable the gel to absorb or carry molecular oxygen and thereby produce a concentration of molecular oxygen in the gel that is higher than a concentration of molecular oxygen that would otherwise be present in a hydrated gel, that lacks the perfluorocarbon compound, solely by absorption of oxygen from the atmosphere at standard temperature and pressure, the higher concentration of molecular oxygen in the hydrated gel allowing the seed analog to exhibit a higher germination rate than exhibited by an otherwise similar control seed analog lacking the higher concentration of molecular oxygen.
2. An analog of a botanic seed as recited in claim 1 wherein the totipotent plant tissue is a plant embryo.
3. An analog of a botanic seed as recited in claim 2 wherein the plant embryo includes a radicle and the gel has a concentration of dissolved oxygen sufficient to support elongation of the radicle out of the gel.
4. An analog of a botanic seed as recited in claim 1 wherein the gel includes plant nutrients.
5. An analog of a botanic seed as recited in claim 1 wherein the gel comprises complexed sodium alginate.
6. An analog of a botanic seed as recited in claim 5 wherein the sodium alginate is present at a concentration within a range of about 0.5% w/v to about 2.5% w/v.
7. An analog of a botanic seed as recited in claim 6 wherein the sodium alginate concentration is within a range of about 0.9% w/v to 1.5% w/v.
8. An analog of a botanic seed as recited in claim 1 wherein the gel comprises agar.
9. An analog of a botanic seed as recited in claim 8 wherein agar is present in the gel at a concentration within a range of about 5% w/v to about 10% w/v.
10. An analog of a botanic seed as recited in claim 1 including a rigid shell in surrounding relationship to the gel.
11. An analog of a botanic seed comprising:a unit of plant embryonic tissue; and a substantially non-phytotoxic hydrated gel in surrounding relationship to the unit of plant embryonic tissue, the hydrated gel including a perfluorocarbon compound that confers on the hydrated gel an ability to absorb a greater amount of molecular oxygen from the atmosphere than would otherwise be absorbable by the hydrated gel, lacking the perfluorocarbon compound, from the atmosphere at standard temperature and pressure, the greater amount of absorbed molecular oxygen allowing the seed analog to exhibit a germination rate that is higher than exhibited by an otherwise similar control seed analog lacking the perfluorocarbon compound. 12. An analog of a botanic seed as recited in claim 11 wherein the gel further comprises plant nutrients.
13. An analog of a botanic seed comprising:a unit of plant embryonic tissue; a substantially non-phytotoxic hydrated gel in surrounding relationship to the unit of plant embryonic tissue, the hydrated gel including plant nutrients and at least one perfluorocarbon compound, the perfluorocarbon compound conferring on the hydrated gel an ability to absorb a greater amount of molecular oxygen from the atmosphere than would otherwise be absorbable by said hydrated gel, lacking the perfluorocarbon compound, from the atmosphere at standard temperature and pressure, the greater amount of absorbed molecular oxygen allowing the seed analog to exhibit a germination rate that is higher than exhibited by an otherwise similar control seed analog lacking the perfluorocarbon compound; and a rigid outer shell in surrounding relationship to the hydrated gel. 14. An analog of a botanic seed as recited in claim 13 wherein the outer shell comprises a material selected from a group consisting of cellulose, glass, plastic, cured polymeric resins, and combinations thereof.
15. An analog of a botanic seed as recited in claim 13 wherein the outer shell comprises a rigid cellulosic inner layer surrounding the hydrated gel and a polymeric outer layer surrounding the cellulosic inner layer, where the polymeric outer layer has a high dry strength and a low wet strength.
16. A method for germinating unit of totipotent plant tissue comprising:(a) placing a unit of totipotent plant tissue in contact with a unit of non-phytotoxic hydrated gel so as to form a seed analog, the gel comprising a perfluorocarbon compound, the perfluorocarbon compound enabling the gel to acquire from the atmosphere a concentration of molecular oxygen in the gel that is higher than a concentration of molecular oxygen that would be present in a gel, that lacks the perfluorocarbon compound, solely by absorption of oxygen from the atmosphere at standard temperature and pressure, the higher concentration of molecular oxygen in the gel allowing the seed analog to exhibit a higher germination rate than exhibited by an otherwise similar control seed analog lacking the perfluorocarbon compound; and (b) incubating the seed analog under environmental conditions conducive to plant growth so as to cause the totipotent plant tissue to grow and develop into a germinant. 17. A method as recited in claim 16 wherein step (a) comprises encapsulating the unit of totipotent plant tissue in said unit of hydrated oxygenated gel.
18. A method as recited in claim 17 including the step, after step (a) but before step (b), of encapsulating the unit of hydrated oxygenated gel in a rigid outer shell.
19. A method for germinating a unit of totipotent plant tissue comprising:(a) placing a unit of totipotent plant tissue in contact with a unit of hydrated gel comprising a perfluorocarbon compound so as to form a seed analog; (b) exposing the seed analog to a gas having a concentration of oxygen greater than atmospheric oxygen at standard temperature and pressure so as to cause the gel to acquire, by absorption from said gas, a concentration of molecular oxygen that is higher than would otherwise be present in the gel solely by absorption of oxygen from the atmosphere at standard temperature and pressure, the higher concentration of absorbed molecular oxygen in the gel allowing the seed analog to exhibit a higher germination rate than exhibited by an otherwise similar control seed analog lacking the higher concentration of molecular oxygen; and (c) incubating the seed analog under environmental conditions conducive to plant growth so as to cause the totipotent plant tissue to grow and develop into a germinant. 20. A method as recited in claim 19 wherein said gas is substantially saturated with water vapor.
21. A method for germinating a unit of totipotent plant tissue comprising:(a) preparing a hydrated gel that comprises a perfluorocarbon compound; (b) placing a unit of totipotent plant tissue in contact with a unit of the hydrated gel so as to form a seed analog; and (c) incubating the seed analog under environmental conditions conducive to plant growth so as to cause the totipotent plant tissue to grow and develop into a germinant. 22. A method as recited in claim 21 wherein step (a) comprises:adding the oxygen-carrying compound to an aqueous gel solution to produce a liquid gel mixture; and curing the gel mixture. 23. A method as recited in claim 21 wherein step (a) comprises adding an oxygen-carrying substance to an aqueous gel solution to produce a liquid gel mixture, and step (b) comprises covering at least a portion of the totipotent plant tissue with a volume of the liquid gel mixture, then curing the gel mixture.
24. A method for germinating a unit of totipotent plant tissue, comprising:(a) adding a perfluorocarbon emulsion to an aqueous gel solution to produce a liquid gel mixture; (b) curing the gel mixture to form a hydrated gel; (c) placing a unit of totipotent plant tissue in contact with a unit of the hydrated gel so as to form a seed analog; and (d) incubating the seed analog under environmental conditions conducive to plant growth so as to cause the totipotent plant tissue to grow and develop into a germinant. Description
CROSS REFERENCE TO RELATED APPLICATION This application is a continuation of U.S. patent application Ser. No. 07/604,656, filed on Oct. 26, 1990, now U.S. Pat. No. 5,236,469.
Some researchers have experimented with the production of "artificial" seeds in which individual plant somatic or zygotic embryos are encapsulated in a hydrated gel. (As used herein, "hydrated" denotes the presence of free water interspersed throughout the matrix of gel molecules comprising the gel capsule.) This method evolved from other work showing that encapsulating seeds in hydrated gels can improve germination in some species, especially since such gels can be supplemented with plant hormones and other compounds that aid germination and improve seedling survival in the field. With respect to artificial seeds, reference is made to European Patent Application No. 0,107,141 to Plant Genetics, Inc., published on May 2, 1984 (claiming priority under U.S. Pat. No. 4,562,663, filed on Oct. 12, 1982), teaching that hydrated gels used to encapsulate plant embryos should permit gas diffusion from the environment to the embryo and protect the embryo from abrasion. A suitable gel can be selected from alginates, guar gums, agar, agarose, gelatin, starch, polyacrylamide, and other gels. The gel can include additives such as plant nutrients, pesticides, and hormones. If necessary, the gel can be surface-hardened to confer further resistance to abrasion and penetration.
SUMMARY OF THE INVENTION In accordance with the present invention, an analog of botanic seed is provided which comprises a plant embryo or other unit of totipotent plant tissue encapsulated, or at least in contact with, a hydrated oxygenated gel. The gel preferably also includes dissolved nutrients and other beneficial compounds such as vitamins, hormones, and sources of carbon and energy, which can be utilized by the germinating embryo for enhanced growth or improved probability of survival. Suitable gel solutes are substantially non-phytotoxic and can be selected from a number of different types such as, but not limited to, sodium alginate, agar, agarose, amylose, pectins, dextran, gelatin, starch, modified celluloses, and polyacrylamide.
One way of achieving oxygenation is to bubble oxygen gas through a gel solution before curing the gel. Alternatively, gel capsules can be oxygenated by exposure to oxygen, under pressure if necessary, after curing.
The concentration of perfluorocarbon (or silicone oil) can depend on the oxygen requirements of the plant species being encapsulated in the gel, the oxygen-carrying capability of the perfluorocarbon (or silicone oil) being used, the type of gel, or the size of the microdroplets comprising the emulsion. Generally, the concentration of the perfluorocarbon (or silicone oil) in the gel is about 15% w/v or less.
Although the embryo need only contact the hydrated oxygenated gel, such as by resting on a surface of such gel, the embryo is preferably encapsulated in the gel. Encapsulation allows the resulting analog of botanic seed to be handled without the possibility of the embryo losing contact with the gel.
An analog of botanic seed according to the present invention can also include a rigid outer shell for increased protection against desiccation and physical trauma. The outer shell can have a tapered or wedge-shaped end to facilitate emergence of the radicle during germination. The outer shell preferably has an orifice or analogous feature, or readily breaks apart during germination, making it relatively easy for the embryonic radicle to burst from the analog during germination. The outer shell can be fabricated from a variety of materials including, but not limited to, cellulosic materials, glass, plastic, cured polymeric resins, paraffin, and combinations thereof.
A further object is to provide such an analog with an outer shell for increased protection of the gel and embryo from desiccation and physical trauma but which does not impede the supply of oxygen to the embryo while allowing the embryo to burst through the outer layer during germination.
The foregoing objects and other features and advantages of the present invention will be more fully understood as the detailed description thereof proceeds, particularly when considered together with the accompanying drawing.
FIG. 3B is a crossesectional view of an alternative embodiment of the analog of botanic seed shown in FIG. 3A.
FIG. 5 is a stepwise sequential diagram illustrating a second form of germination pattern frequently observed with an analog of botanic seed according to the present invention, wherein the gel capsule remains attached for a time to the germinating embryo at the cotyledon region in a manner similar to that of a natural seed.
DETAILED DESCRIPTION The analog of botanic seed disclosed herein comprises a unit of totipotent plant tissue having at least one surface in contact with a cured, hydrated, oxygenated gel.
Meristematic tissue is comprised of undifferentiated plant cells that divide to yield other meristematic cells as well as differentiated cells that elongate and further specialize to form structural tissues and organs of the plant. Meristematic tissue is located, for example, at the extreme tips of growing shoots or roots, in buds and in the cambium layer of woody plants.
The material used to encapsulate the totipotent plant tissue is a hydrated gel. A "gel" is a substance that is prepared as a colloidal solution and that will, or can be caused to, form a semisolid material.
As used herein, "hydrated" denotes water-containing. Such gels are prepared by first dissolving in water (where water serves as the solvent, or "continuous phase") a hydrophilic polymeric substance (serving as the solute, or "disperse phase") that, upon curing (or "gelling"), combines with the continuous phase to form the semisolid material. In other words, the water becomes homogeneously associated with the solute molecules without experiencing any substantial separation of the continuous phase from the disperse phase. However, water molecules can be freely withdrawn from a cured hydrated gel, such as by evaporation or imbibition by a germinating embryo. When cured, these gels have the familiar characteristic of compliant solids, like a mass of gelatin, where the compliance becomes progressively less and the gel becomes more "solid" to the touch as the relative amount of water in the gel is decreased.
Gels are typically prepared by dissolving a gel solute, usually in fine particulate form, in water to form a gel solution. Depending upon the particular gel solute, heating is usually necessary, sometimes to boiling, before the gel solute will dissolve. Subsequent cooling will cause many gel solutions to reversibly "cure" or become gelled. Examples include gelatin, agar, and agarose. Such gel solutes are termed "reversible" because reheating cured gel will re-form the gel solution. Solutions of other gel solutes require a "complexing" agent which serves to chemically cure the gel by crosslinking gel solute molecules. For example, sodium alginate is cured by adding calcium nitrate (Ca(NO3)2) or salts of other divalent ions such as, but not limited to, calcium, barium, lead, copper, strontium, cadmium, zinc, nickel, cobalt, magnesium, and iron to the gel solution. Many of the gel solutes requiring complexing agents become irreversibly cured, where reheating will not re-establish the gel solution.
The concentration of gel solute required to prepare a satisfactory gel for encapsulation purposes according to the present invention varies depending upon the particular gel solute. For example, a useful concentration of sodium alginate is within a range of about 0.5% w/v to about 2.5% w/v, preferably about 0.9% w/v to 1.5% w/v. A useful concentration of agar is within a range of about 5% w/v to about 10% w/v, preferably about 8% w/v. (As used herein, the "% w/v" concentration unit is equivalent to grams of solute per 100 ml of solvent.) Gel concentrations up to about 24% w/v have been successfully employed for other gels. In general, gels cured by complexing require less gel solute to form a satisfactory gel than "reversible" gels.
It is preferable to provide the embryo with the usual spectrum of plant nutrients and other beneficial substances such as vitamins and a source of carbon and energy (herein collectively termed generally "nutrients") while the embryo is encapsulated in the gel. Typical ways of providing nutrients are to dissolve the gel solute in a solution of the nutrients or to add a volume of concentrated nutrient solution to the gel solution before curing the gel. In this way, when the gel cures, any areas of the embryo in contact with the gel are also in direct contact with nutrient solutes, where the nutrient solutes are present in substantially uniform concentrations throughout the gel. Another way to provide nutrients is to place a gel capsule containing the embryo but lacking nutrients in contact with a second mass of the same or a different type of hydrated gel which does contain nutrients. As a result of a nutrient concentration gradient between the two hydrated gel masses, nutrients will migrate from the nutrient-containing gel mass to the embryo-containing gel mass.
______________________________________NH4 NO3 1650    mg/LKNO3         1900    mg/LCaCl2.2H2 O             440     mg/LMgSO4.7H2 O             370     mg/LKH2 PO4 170     mg/LNa2 EDTA     37.25   mg/LFeSO4.7H2 O             27.85   mg/LMnSO4.4H2 O             22.3    mg/LZnSO4.4H2 O             8.6     mg/LH3 BO3  6.2     mg/LKI                0.83    mg/LNa2 MoO4.2H2 O             0.25    mg/LCuSO4.5H2 O             0.025   mg/LCoCl2.6H2 O             0.025   mg/LGlycine           0.2     mg/100 cm3Nicotinic Acid    0.05    mg/100 cm3Pyridoxine.HCl    0.05    mg/100 cm3Thiamine.HCl      0.01    mg/100 cm3Kinetin           0.1     mg/LMyo-inositol      100     mg/LIAA               10      mg/LSucrose           30000   mg/LpH                5.7-5.8______________________________________
As used herein, an "oxygenated" gel has a concentration of oxygen therein that is higher than the concentration of oxygen at standard temperature and pressure that would be present in the gel as a result only of absorption from the atmosphere. An "oxygen-carrying" gel as used herein is one that has any extraneously-added oxygen-absorbing or oxygen-carrying substances.
Oxygenation of a gel can be achieved by several methods. First, a gel solution can be oxygenated before curing by passing oxygen gas through the solution. On a laboratory scale, this is typically performed by placing the solution in a "gas-washing bottle" known in the art and bubbling oxygen gas through the solution while the solution is in the bottle. Analogous methods can be employed for oxygenation of large volumes and for oxygenation of a continuous stream of uncured gel. When oxygenating a gel solution in this manner, it should be kept in mind that hot solutions generally absorb less oxygen than cold solutions. Second, a gel can be oxygenated after curing by, for example, placing the gel in a pressurized oxygen or pure oxygen environment. These methods are also effective when the gel contains oxygen-carrier or oxygen-absorbing compounds, discussed in further detail below.
The concentration of oxygen in an oxygenated gel will depend on a number of factors. In terms of a lower threshold concentration, the oxygen concentration in a gel capsule surrounding an embryo is preferably at least adequate to support enough growth of the radicle (embryonic structure that eventually becomes the plant root) for it to rupture the capsule and become exposed to oxygen in the atmosphere. The radicle is very sensitive to oxygen concentration. For example, if the oxygen concentration in a gel capsule surrounding an embryo is too low, the meristematic tissue in the radicle dies before the radicle can grow out of the capsule (see Example 2). Generally, if the oxygen concentration is high enough for growth of the radicle, it is also high enough to support protrusive growth of other parts of the plant embryo from the capsule, such as the shoot. The lower threshold concentration of oxygen seems to depend in part on the particular plant species represented by the embryo. Other determinants of the concentration of oxygen in a gel include the thickness of the gel, the fact that different types of gel solutes will absorb different amounts of oxygen, the degree of hydration of the gel, the concentration of the gel solute, presence or absence of other solutes in the gel such as nutrients and concentrations thereof, and the temperature of the gel. Therefore, in most cases, the lower threshold oxygen concentration is best determined for a specific plant embryo and capsule configuration by performing a simple germination experiment involving a series of identically encapsulated embryos in which each gel capsule in the series has a stepwise different oxygen concentration from all other capsules in the series.
A preferred class of compounds for use in increasing the concentration of oxygen in a gel are the perfluorocarbons (PFCs). These compounds are organic compounds in which all hydrogen atoms have been replaced by fluorine atoms. They are nonpolar, colorless, odorless, non-toxic, heat-stable, and extremely chemically inert. Because gases such as carbon dioxide and oxygen have a high solubility in PFCs, PFC compounds have been studied for use as blood substitutes. A first representative group of suitable PFCs comprises the perfluorocycloalkanes and perfluoro(alkylcycloalkanes) such as perfluorodecalin. A second representative group comprises the perfluoro(alkylsaturated heterocyclic) compounds such as perfluorobutyltetrahydrofuran. A third representative group comprises the perfluoro(tert-amine) compounds such as perfluorotributylamine.
The maximum amount of surfactant required to achieve a suitably stabilized emulsion is generally about 10% w/v, but can be higher if extremely small microdroplets of PFC are formed during emulsification. In other words, as the diameter of microdroplets in a unit volume of PFC emulsion is decreased, the surface area of the PFC disperse phase is increased, and a correspondingly greater amount of surfactant is required to suitably stabilize the emulsion. The preferred range of surfactant concentration is from about 0.4% w/v to about 6% w/v. The surfactant is typically added to a suspension of uncured gel and PFC just before creation of the emulsion.
An alternative oxygen-absorbing compound that can be incorporated as an emulsion into a hydrated gel is σ silicone oil. Silicone oils are available in a number of viscosity values, where oils having a viscosity within the range of about 0.65 to about 15 centipoise are preferred. These oils, like PFCs, are nonpolar, colorless, odorless, non-toxic, heat-stable, chemically inert, and have high oxygen solubility values. In fact, some silicone oils have higher oxygen solubilities than many PFCs. Emulsifying a silicone oil in a gel solution is performed in substantially the same way as emulsifying a PFC. As with PFCs, a surfactant is generally required to achieve a suitably stable emulsion of silicone oil. Also, the concentration of silicone oil in a gel is generally about 25% w/v or less.
After preparing the gel liquid, whether it includes emulsified PFC or silicone oil or not, preparing units of cured gel for use in germinating plant embryos can be done in a number of ways. The method chosen will depend in part upon how the embryo will contact the gel. It is important that the embryo have contact with the gel, either directly or via an intervening water-permeable "bridge" such as filter paper. In general, the embryo can rest on a surface of an oxygenated gel, rest in a preformed hole in a block of gel, or be entirely encapsulated in the gel. In the first two methods, the gel is generally cured preformed into the preferred shape, or can be formed as a larger cured mass and cut to size before inserting the embryo. In the case of totally encapsulating an embryo in the gel, it is preferable to insert the embryo in a unit of gel having the desired volume before the gel is completely cured.
FIG. 1A is a cross-sectional view of an analog of a botanic seed 10 made by totally encapsulating an embryo 12 in a hydrated oxygenated gel capsule 14. One way to make such a capsule is to place the uncured gel mixture in a separatory funnel. The stopcock on the funnel is adjusted to form drops of the gel liquid in a slow stepwise manner. Whenever a drop forms at the tip of the separatory funnel, an embryo is inserted fully into the drop using sterile forceps. Then, the drop containing the embryo is either captured in a space conforming to the desired shape of the capsule for curing or, in the case of gels that must be complexed to cure, dropped into complexing solution until curing is complete.
FIG. 1B is a cross-sectional view of an analog of a botanic seed 20 where a large portion 22 of the gel capsule is preformed. In FIG. 1B, the large portion 22 is shown in the shape of a cube, although other shapes will also suffice, such as spherical or ovoid. The larger portion 22 has a bore 24, which can also be preformed or cut after forming, into which the embryo 12 is inserted. If desired, the bore 24 can be sealed with a plug 26 after inserting the embryo 12. The plug 26 can be made of an additional piece of cured gel or other suitable material such as paraffin or similar material.
As can be seen in FIG. 1C, it is also possible to make an analog of botanic seed 30 by preforming two opposing capsule halves 32a, 32b which, when pressed together to form a complete capsule 34, define a cavity 36 for receiving the embryo 12. Again, although FIG. 1C shows a cubic configuration, the general concept shown therein is adaptable to a variety of shapes.
It is readily ascertainable that variations on each of the three embodiments shown in FIGS. 1A, 1B, and 1C can be made which are within the scope of an encapsulated embryo according to the present invention.
It is also readily apparent that the embodiments of FIGS. 1A, 1B, and 1C can be made via an automated process.
It is also possible to encase an analog of botanic seed comprising an embryo-containing gel capsule in a rigid shell to afford protection to the gel capsule and to the embryo therein from physical injury, desiccation, and other adverse environmental forces. For example, FIG. 2A shows a cross-sectional view of one embodiment of such an analog 40 comprising an embryo 12, a capsule 42 comprised of a hydrated oxygenated gel in surrounding relationship to the embryo 12, and an outer shell 44 in surrounding relationship to the gel capsule 42. The outer shell 44 can be made from a large variety of materials including, but not limited to, a cellulosic material, paraffin, moldable plastic or cured polymeric resin, or a combination of these and/or other materials characterized by non-toxicity and suitable rigidity. However, the rigidity must not be such that an embryo germinating from within would not be capable of growing out of the analog 40 without fatal or debilitating injury. Hence, polymeric materials having a high dry strength and low wet strength are particularly desirable. Also desirable are shell materials that break apart easily upon application of an outwardly protrusive force from inside the capsule but are relatively resistant to compressive forces applied to the outside of the capsule. The outer shell 44 preferably also has an opening 46 toward which the radicle 48 of the embryo 12 is oriented so as to facilitate protrusive growth of the radicle 48 from the analog 40 during germination. Otherwise, the radicle could become trapped inside the analog 40 and be prevented from successfully germinating.
Another possible embodiment of a shell-encased embryo-containing gel capsule is illustrated in FIG. 2B showing a cross-sectional view of an analog of botanic seed 50. The analog 50 comprises an embryo 12 and a capsule 52 comprised of a hydrated oxygenated gel in surrounding relationship to the embryo 12, where the capsule 52 is cast in an inner shell 54 to create a particular shape, such as a cylinder. The inner shell 54 can be cut, for example, from a plastic or cellulosic drinking straw or analogous material such as glass tubing. Then, the capsule-containing inner shell 54 is coated or otherwise layered with an outer shell 56 similar to the outer shell 44 of FIG. 2A. Again, it is preferable that the outer shell 56 include an opening 58 to ease protrusion of the germinating radicle. It is also preferable that the outer shell 56 have a low wet strength and a high dry strength.
Yet another possible embodiment of a shell-encased embryo-containing gel capsule is illustrated in FIG. 2C showing a cross-sectional view of an analog of botanic seed 60. As in FIG. 2B, the analog 60 in FIG. 2C comprises an embryo 12, a capsule 52 comprised of a hydrated oxygenated gel in surrounding relationship to the embryo 12, and a rigid cylindrical shell 62 similar to the inner shell 54 of FIG. 2B. In addition, a cap 64 of paraffin or other polymeric material is applied to at least the first end 66 to afford protection against desiccation and physical trauma as well as to properly restrain the cotyledons to facilitate normal germination. A second cap (not shown) similar to the first cap 64 can also be applied to the second end 68 for additional protection. If the shell 62 is made from a water-impermeable substance, it is preferable that the cap 64, especially if applied to both ends 66, 68, be made from a water-permeable substance to ensure adequate water penetration to the embryo 12 to support germination.
In addition, whenever an embryo-containing gel capsule is substantially surrounded by an outer shell, it is at least partially isolated from the atmosphere. As a result, the gel should contain an emulsion as described above and be oxygen-charged to ensure that a sufficient supply of oxygen is present in the gel to supply the needs of the embryo during germination.
The embodiments shown in FIGS. 1A-1C and FIGS. 2A-2C are merely representative examples of possible capsule geometries. Other geometries and capsule configurations are possible. For example, FIGS. 3A-3C show cross-sectional views of three other embodiments wherein the capsules are bullet-shaped. Although capsules having such a shape can be useful for mechanical sowing, that is not the principal intent of the bullet shape. Rather, a tapered "bullet" end of a capsule helps guide an embryonic radicle germinating from within the capsule to grow toward the "bullet" apex for ease of escape from the capsule. As with natural seeds, the capsules can be sown in any orientation in a soil or the like without interfering with the normal geotropism of the radicle.
FIG. 3A also shows an outer shell 75 in surrounding relationship to the block 71 and nutrient unit 74 as well as the embryo 12. To permit use of commonly available materials as the outer shell 75, such as tubular materials, the outer shell 75 preferably has a circular transverse cross-section, giving the outer shell 75 a cylindrical shape with a tapered first end 73 and a second end 76. The outer shell 75 can be constructed of, for example, a cellulosic tubular material similar to a paper drinking straw. Other materials such as plastic are also suitable. The tapered first end 73 can be formed via radicle crimps 77 or other constriction method to reduce the diameter of the outer shell 75 at the tapered first end 73. The second end 76 can be similarly tapered (not shown) or it can be shaped as shown as a transverse circular flat contiguous with the outer shell 75. The tapered first end 73 preferably terminates with an orifice 78 toward which the radicle 48 is urged to grow by the tapered first end 73 during germination. If required, the orifice 78 can be occluded with a covering 79 comprised of a soft material such as paraffin or, preferably, any suitable material having a high dry strength and a low wet strength. Alternatively, the covering 79 can be comprised of a material that breaks apart easily upon application of a protrusive force from inside the capsule.
FIGS. 4 and 5 each show stepwise sequential images of a gymnosperm embryo 12 germinating from an analog of botanic seed 100. Although the analog 100 is shown comprising an ovoid-shaped hydrated oxygenated gel capsule 101, FIGS. 4 and 5 are also applicable to other capsule embodiments, such as those including an outer shell. For simplicity, the analog 100 is shown being "sown" by placing on top of a soil surface 102, even though in most cases the analog 100 would be sown beneath the soil surface 102. Also, for clarity, each image except the rightmost image in each of FIGS. 4 and 5 is shown as a cross-sectional view.
For purposes of comparison, FIG. 5 shows a germination pattern closely resembling that of a natural seed. In the first, or leftmost, image, an analog of botanic seed 100 is comprised of an embryo 12, having a radicle 48 and cotyledons 49, and a hydrogenated oxygenated gel capsule 101 in surrounding relationship to the embryo 12. In the second image, the radicle 132 has burst from the capsule 134. In the third image, a root 136 is shown penetrating the soil surface 102 and the cotyledons 138 have elongated. The capsule 140 has a certain strength, such as a surface strength, sufficient to prevent the cotyledons 138 from rupturing the capsule 140 during elongation while allowing the capsule 140 to be pushed ahead of the growing cotyledons 138. In the fourth image, the root 142 and cotyledons 144 have grown longer. The capsule 146 remains attached to the cotyledons 144 while allowing them to elongate naturally without malforming. In the fifth image, the seedling 148 has elongated sufficiently to elevate the capsule 150 off the soil surface 102. Finally, in the rightmost image, the capsule 152 has fallen off the cotyledons 154 in a manner similar to a seed husk of a natural seed. The seedling 156 appears normal and has excellent prospects for future growth.
In the Examples below, a growth pattern such as that shown in FIG. 4 wherein the capsule remains adhered to the hypocotyl of a germinated embryo for a time is regarded as not as desirable as that shown in FIG. 5 wherein the capsule remains attached for a time to the cotyledons in a manner similar to a natural seed. Nevertheless, there is no evidence that a germination pattern as in FIG. 4 is in any way detrimental to the survival of the seedling. The germination patterns discussed above in relation to FIGS. 4 and 5 have been regularly observed during numerous studies of various embodiments of analogs of botanic seed according to the present invention. While the pattern of FIG. 5 more closely resembles that of a germinating natural seed, both the FIG. 4 and FIG. 5 patterns will result in production of normal seedlings.
"Hypocotyl length" pertains to the length of the hypocotyl at the time the hypocotyl was measured,
EXAMPLE 1 This Example is an evaluation, for comparison purposes, of embryo germination from non-oxygenated capsules of the type as disclosed in European Patent Application No. 0,107,141. (The European application referred to herein as EPA '141 claims priority under U.S. Pat. No. 4,562,663, filed on Oct. 12, 1982.) Individual sets of zygotic embryos of Norway Spruce were subjected to one of the following Treatments:
TABLE I______________________________________    Normal      Mean Length                           Mean LengthTreatment    Germinants  Hypocotyls Radicles______________________________________1 (Control)    81%         1.26 cm    1.44 cm2 (Offset)    17%         0.63 cm    0.98 cm3 (Centered)     8%         0.62 cm    0.72 cm______________________________________
Treatment (1): "Control" wherein bare embryos placed on the surface of nutrient agar in a manner known in the art.
Treatment (4): Embryos encapsulated in blocks of nutrient agar (0.8%) with radicles protruding from the block: blocks then placed on the surface of nutrient agar.
Treatment (5): Embryos encapsulated as in EPA '141 except that the alginate concentration was 1.5%, and a nutrient aqueous liquid containing dissolved nutrients as in "MS liquid" was used instead of water to dissolve the alginate: capsule diameter was about 3 mm: capsules then placed on the surface of nutrient agar.
To prepare agar blocks for Treatments (3) and (4), blocks of nutrient-containing agar were cut measuring about 4�4�5 mm using a small spatula. Using sterile forceps, an embryo was inserted into each block, centered in the block for Treatment (3) and with the radicle protruding outside the block for Treatment (4). Embryos were inserted into the blocks radicle-end first for Treatment (3) and cotyledon-end first for Treatment (4). With Treatment (4), about half the embryo length was left protruding from the agar block.
TABLE II______________________________________       %       Normal   % Germinants                            % Germinants       Germi-   w/Swollen   w/swollenTreatment   nants    Hypocotyls  Cotyledons______________________________________1 (Ager control)       90%       0%         0%2 (Alginate control)        8%      36%         0%3            0%      91%         47%4           61%      37%         26%5           20%      75%         3%______________________________________
Treatment (2): As shown in FIG. 6, glass cylindrical capsule shells 160 were made having length about 12 mm, outside diameter about 7 mm, and inside diameter about 5.6 mm. One end 161 of each shell was sealed with an elastomeric septum 162. After sterilization, the shells were oriented vertically open-end up and filled about two-thirds full with nutrient agar medium 163. A zygotic embryo 164 was inserted halfway into the exposed agar surface 165 in each shell, cotyledon end 166 first, leaving the radicle 167 exposed to the atmosphere. The resulting capsules 168 were turned on their sides on a nutrient agar surface for incubation.
Treatment (3): Same as Treatment (2) except that, after inserting the embryos in the agar, the open ends of the glass shells were subsequently partially sealed from the atmosphere using PARAFILM (a registered trademark of American National Can, Greenwich, Conn.) laboratory film (a paraffin film well-known in the art). The film was applied to the open end in a manner that left a small hole through which the radicle could protrude during germination.
TABLE III__________________________________________________________________________                   % Embryos % Normal       % Swollen             % Swollen                   Completely                          % CotyledonsTreatment Germinants       Hypocotyls             Cotyledons                   Trapped                          Trapped__________________________________________________________________________1 (Control) 72%   22%   6%     0%     0%2     39%   44%   11%    6%    78%3      6%   78%   45%   28%    61%4     72%   17%   0%    17%    61%5     12%   45%   0%    39%    61%6     50%   34%   6%     6%    61%7     34%   45%   6%    11%    79%__________________________________________________________________________
Conclusions based on Table III and other observations were summarized as follows:
(a) All Treatments lacking the PARAFILM-sealed end (Treatments (1), (2), (4), and (6)) exhibited a higher percent of normal germination, indicating a benefit of free exposure of the embryos to oxygen.
(b) Controls (Treatment (1)) as well as Treatment (4) exhibited the highest percentages of normal germinants (72%), followed by Treatment (6) at 50% and Treatment (2) at 39%. Apparently, the combination of light capsule weight and exposure of at least the radicle to oxygen during germination was beneficial.
(c) No swollen cotyledons were seen in embryos that experienced Treatment (4) or Treatment (5), indicating a benefit of lightweight capsules.
(d) Treatments (3)-(6) exhibited the same percent of trapped cotyledons, even though the amount of medium in the capsules differed between Treatment (3), Treatments (4) and (5), and Treatment (6). Apparently, these capsule geometries are not optimal for allowing early release therefrom during gymnosperm germination.
(e) Partially sealing the radicle-end of the capsules with PARAFILM resulted in lower average lengths of hypocotyls and radicles (data not shown), probably demonstrating a slight negative effect of partial (although not excessive) physical obstruction of the radicle until it penetrated the opening in the PARAFILM.
(f) Treatment (4) embryos experienced the same percent normal germinants as the controls of Treatment (1). However, average lengths of hypocotyls and radicles, as well as average seedling weights (data not shown) of Treatment (4) embryos, were less than with control embryos. Such decreased values, however, probably merely reflect the slightly greater physical restraints placed on a Treatment (4) embryo versus a "bare" embryo when undergoing germination.
EXAMPLE 5 In this Example, we evaluated enclosing the embryo in a porous tube embedded in a nutrient-containing gel as an improved means for physically securing an embryo inside a gel capsule without actually embedding an embryo directly in the gel. This method was investigated because "shelf" capsules such as described in Treatment (4) of Example 4 generally cannot be turned or handled without the embryo falling off the gel "shelf." The capsules tested in this Example also included a rigid exterior shell for additional physical protection. Securing the embryo was performed using a tube made of filter paper, where the filter paper served as a liquid "bridge" between the gel and the embryo.
Individual sets of Norway Spruce embryos were subjected to one of the following Treatments:
Treatment (1): "Control" as in Treatment (1) of Example 4.
Treatment (2): As in Treatment (4) of Example 4.
Treatment (3): Glass shells having 5.2 mm inside diameter were made as described in Treatment (2) of Example 4. One end of each shell was sealed with an elastomeric septum, then the shells were sterilized. Each shell was then filled with nutrient agar. Small paper tubes having 2.5 mm inside diameter and about 5 mm long were made by cutting Whatman #1 qualitative filter paper into 5 mm-wide strips, each of which was rolled around a 2.5 mm outside diameter pin to form a paper tube. The tubes were kept from uncurling by application of a small piece of label tape (2�8 mm). The tubes were autoclaved and sealed on one end by dipping in hot paraffin. Each tube was axially inserted sealed-end first in an individual agar-containing glass shell until the open end of the tube was flush with the opening of the shell. An embryo was inserted in each paper tube cotyledon-end first until the radicle tip was flush with the tube opening.
Treatment (4): Same as Treatment (3) except that the paper tubes were 3.6 mm inside diameter instead of 2.5 mm inside diameter.
Each Treatment involved six sets having six embryos per set. In Treatments (2)-(4), the resulting capsules were placed on their sides on nutrient agar surfaces in sterile covered Petri plates and incubated under continuous light for 35 days at 23� C. Data are tabulated in Table IV.
TABLE IV______________________________________                           % Trapped   Normal     Trapped      But NormalTreatment   Germinants Cotyledons (All)                           Cotyledons______________________________________1 (Control)   91%        --           --2       86%        92%          85%3       20%        87%          19%4       33%        75%          16%______________________________________
(b) In Treatments (2) to (4) involving encapsulation of the embryos, the cotyledons of a large percentage of germinants remained in the capsule after five weeks' incubation. This did not adversely affect normalcy in Treatment (2), but did in Treatments (3) and (4).
Treatment (2): A 1:1 v/v mixture of the 30% FC-77 emulsion with 2�-concentrated nutrient liquid containing alginate.
Treatment (3): A 2:1 v/v mixture of the 30% FC-77 emulsion and 3�-concentrated nutrient liquid containing alginate.
Treatment (4): A 3:1 v/v mixture of the 30% FC-77 emulsion and 4�-concentrated nutrient liquid containing alginate.
Treatment (5): A 4:1 v/v mixture of the 30% FC-77 emulsion and 5�-concentrated nutrient liquid containing alginate.
Treatment (6): "Control"; bare embryo placed on 1�-concentrated nutrient liquid containing agar.
For Treatments (1)-(5), the mixtures of emulsion and nutrient liquid with alginate were transferred immediately after preparation to a sterile gas-washing bottle and oxygenated using sterile oxygen passing therethrough for 30 minutes. The oxygenated mixtures were then placed individually in a separatory funnel. Embryos were encapsulated in a manner similar to that disclosed in EPA '141 using 100 mM Ca(NO3)2 for complexing and nutrient liquid for rinsing. After encapsulation, the capsules were placed on the surface of nutrient agar in covered Petri plates. For each Treatment, three plates were prepared, each containing six embryos. All Treatments were incubated in continuous light at room temperature. A preliminary normalcy evaluation was made after two weeks' incubation and a final evaluation conducted after five weeks.
TABLE V______________________________________             2 Week %   2 Week %  Growing   5 Week %                               5 Week %   Normal    Thru      Normal  GrowingTreatment   Germinants             Capsule   Germinants                               Thru Capsule______________________________________1       6%        34%        0%     17%2       0%        28%       12%     89%3       0%        61%        0%     67%4       12%       56%       28%     50%5       6%        62%       23%     78%6 (Control)   84%       --        88%     --______________________________________
(d) After two weeks' incubation, the Control embryos (Treatment (6)) had the longest mean radicle length, as shown in FIG. 9C. Treatments (1)-(5) had somewhat variable radicle lengths. After five weeks, mean radicle length in the Control was still the longest, but mean lengths in Treatments (1)-(5) were substantially equal to each other, as shown in FIG. 9F. The better growth of radicles after two weeks in Treatments containing higher amounts of FC-77 correlates with the importance of the oxygen supply for radicle growth. The substantially equal growth of radicles in Treatments (3)-(5) indicates that there is a concentration of oxygen in a hydrated gel above which further improvement in radicle growth is not observed. However, as shown in the five week data of FIG. 9F, radicle growth is not permanently inhibited at lower oxygen levels. Once the radicle grows out of an oxygen-limiting environment (i.e., the gel capsule), growth appears to accelerate.
To produce capsules around plant embryos, the oxygenated gel suspension was transferred to a sterile separatory funnel. The stopcock on the separatory funnel was adjusted to form drops in a slow stepwise manner. Whenever a drop of the gel suspension formed at the tip of the separatory funnel, a plant embryo was inserted into the drop using sterile forceps, with the cotyledons pointing upward. The embryo was fully immersed within the drop. The drop was then placed in a solution of 100 mM Ca(NO3)2 with nutrients. This solution, termed a "complexing solution," was adjusted to pH 5.7 and autoclaved prior to use. The capsules were allowed to harden in the calcium nitrate solution for 20 minutes. Then, the calcium nitrate solution was discarded and the capsules rinsed for five minutes with nutrient liquid before placement of the resulting capsules on the surface of nutrient agar in sterile covered Petri plates.
Alginate solution lacking the PFC emulsion was prepared by combining one liter of nutrient liquid with 15 g of Protanal LF-60 alginate. After autoclaving, the gel solution was oxygenated using a gas-washing bottle as described above (if required) and transferred to a sterile separatory funnel. Plant embryos were encapsulated in the alginate as described above.
All Treatments were incubated in continuous light at room temperature for five weeks, at which time they were ,examined for germination and seedling development. The data are shown in Table VI and in FIGS. 10A and 10B.
TABLE VI__________________________________________________________________________  % Normal         % That Grew                 % Radicle                        % Hypocotyl                               % GerminationTreatment  Germinants         Thru Capsule                 Germination                        Germination                               Hyp. &amp; Rad.__________________________________________________________________________ 1 (Control)  92%    --      --     --     -- 2      7%    28%     17%    92%    17% 3     17%    37%     45%    97%    45% 4     46%    87%     87%    100%   87% 5 (Control)  88%    --      --     --     -- 6      3%    24%     21%    100%   21% 7      9%    24%     56%    94%    56% 8     30%    55%     59%    92%    59% 9 (Control)  92%    --      --     --     --10      9%    37%     40%    95%    40%11      3%    12%     15%    54%    15%12     32%    70%     71%    98%    71%13 (Control)  32%    --      --     --     --14      3%    28%     30%    100%   30%15     10%    34%     35%    100%   35%16     21%    42%     47%    100%   47%__________________________________________________________________________
The methods used in this Example were substantially the same as used in Example 7 except that other surfactants and surfactant concentrations were used, The study comprised six Treatments, as follows:
TABLE VII__________________________________________________________________________ % Normal        % Growth               % Radicle                      % Hypocotyl                             % GerminationTreatment Germinants        Thru Capsule               Germination                      Germination                             Hyp. &amp; Rad.__________________________________________________________________________1     56%    94%    94%    100%   94%2     70%    86%    86%     97%   86%3      0%     0%     0%     0%     0%4     15%    57%    59%    100%   59%5     24%    35%    41%     89%   35%6 (Control) 100%   --     --     --     --__________________________________________________________________________
Conclusions drawn from the results can be summarized as follows:
(a) As shown in Table VII, Treatment (3), sodium dodecyl sulfate is not an effective surfactant in that it caused mortality of all embryos in contact with it.
(b) As shown in Table VII, Treatments (2) and (1), respectively, egg albumin and Pluronic F-68 are both effective surfactants for PFCs such as FC-77. Egg albumin produced more normal germinants, but Pluronic F-68 yielded more germinated embryos.
(c) As shown in Table VII and FIG. 11A, oxygenated PFC-containing alginate capsules yielded a higher level of normalcy and a higher total number of germinants than seen with alginate capsules lacking PFC, whether oxygenated or not.
(d) As expected, bare embryos grown on agar produced the most normal germinants.
(e) Treatment (1) yielded the most embryos that grew through the capsule (Table VII).
EXAMPLE 9 In this Example, the ability of various perfluorocarbons to supply oxygen to encapsulated embryos was evaluated.
The methods employed in this Example are the same as those in Example 7 except that several different perfluorocarbons were used. The various Treatments tested were as follows:
Treatment (1): Norway Spruce embryos encapsulated in oxygenated alginate containing an emulsion of 30% FC-77 plus 1.5% Pluronic F-68.
Treatment (2): Norway Spruce embryos encapsulated in oxygenated alginate containing an emulsion of 30% perfluorodecalin (another type of PFC) and 1.5% Pluronic F-68.
Treatment (3): Norway Spruce embryos encapsulated in oxygenated alginate containing an emulsion of 30% perfluorotributylamine (another type of PFC) and 1.5% Pluronic F-68.
Treatment (4): Norway Spruce embryos encapsulated in oxygenated alginate lacking PFC.
Treatment (5): Norway Spruce embryos encapsulated in non-oxygenated alginate lacking PFC.
All Treatments utilized Norway Spruce zygotic embryos and each consisted of six covered Petri plates containing six encapsulated embryos per plate. Treatments were incubated in continuous light at room temperature for five weeks, after which germination success and other parameters were evaluated. Results are tabulated in Table VIII and shown in FIGS. 12A and 12B.
TABLE VIII__________________________________________________________________________ % Normal        % Growth               % Radicle                      % Hypocotyl                             % GerminationTreatment Germinants        Thru Capsule               Germination                      Germination                             Hyp. &amp; Rad.__________________________________________________________________________1     69%    92%    95%    100%   97%2     35%    70%    77%    100%   77%3     61%    81%    86%    100%   56%4     34%    56%    56%    100%   56%5     29%    60%    65%    100%   65%6 (Control) 97%    --     --     --     --__________________________________________________________________________
Treatment (4): Embryos encapsulated in nonoxygenated alginate lacking PFC but containing 1.5% Pluronic F-68.
Treatment (5): Embryos encapsulated in nonoxygenated alginate lacking both PFC and-surfactant.
TABLE IX__________________________________________________________________________ % Normal        % Growth               % Radicle                      % Hypocotyl                             % GerminationTreatment Germinants        Thru Capsule               Germination                      Germination                             Hyp. &amp; Rad.__________________________________________________________________________1     35%    82%    82%    100%   82%2     27%    52%    49%    100%   49%3     18%    36%    36%     97%   36%4      6%    19%    20%     95%   20%5      9%    28%    28%    100%   28%6      6%    34%    34%    100%   34%7 (Control) 94%    --     --     --     --__________________________________________________________________________
(a) As shown in Table IX, both oxygenated and non-oxygenated PFC-containing alginate capsules (Treatments (1) and (2)) yielded more germinants and a higher percent of normal germinant than non-PFC containing alginate capsules.
(b) As shown in Table IX, Pluronic F-68, in an alginate capsule lacking PFC, appears to increase germination when the capsule has been oxygenated (Treatment (3)), and to decrease germination when the capsule is non-oxygenated (Treatment (4)).
(c) Of the capsule formulations tested, the oxygenated alginate capsule containing PFC emulsion appears to be the best.
(d) It appears that the benefit of adding an emulsion of PFC to the alginate capsule is derived from the presence of the PFC and not merely the surfactant therein.
(e) Treatment (1) exhibited the highest percent of embryos that grew through the capsule (Table IX).
(f) Hypocotyl germination was high with all Treatments (Table IX). Treatment (1) exhibited the highest values of percent germination of both radicle and hypocotyl.
(g) The only types of malformations observed were swollen hypocotyls and twisted cotyledons (FIG. 13A).
(h) The controls (Treatment (7)) exhibited the longest radicles and hypocotyls (FIG. 13B). Treatment (1) embryos exhibited the longest hypocotyl lengths of the encapsulated embryos, as well as the longest radicle lengths.
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS3545129 *Jun 20, 1968Dec 8, 1970Canadian Patents DevManufacture of dormant pelleted seedsUS3688437 *Jan 22, 1971Sep 5, 1972Forenode Superfosfatfabrika AbPellets in the form of foamed bodies, and methods for the preparation thereofUS3734987 *Jan 22, 1971May 22, 1973Forenade Superfosfatfabriker AGel capsules for small units and methods of encapsulating such unitsUS3850753 *Mar 13, 1973Nov 26, 1974Tanabe Seiyaku CoCultivation of aerobic microorganismsUS4166006 *Nov 10, 1977Aug 28, 1979Corning Glass WorksMeans for stimulating microbial growthUS4252827 *May 23, 1979Feb 24, 1981The Green Cross CorporationPerfluorodecalin and perfluorotripropylamine; ethylene oxide-propylene oxide copolymer; phospholipid; fatty acid, salt or monoglycerideUS4465017 *Mar 9, 1983Aug 14, 1984Simmons John JSeed coating machineUS4562663 *Oct 12, 1982Jan 7, 1986Plant Genetics, Inc.Analogs of botanic seedUS4583320 *Oct 25, 1983Apr 22, 1986Plant Genetics, Inc.To an environment for growth and developmentUS4615141 *Aug 14, 1984Oct 7, 1986Purdue Research FoundationProcess for encapsulating asexual plant embryosUS4665648 *Jan 28, 1986May 19, 1987Seppic SaFilm-forming compositions for enveloping grains and seedsUS4715143 *Apr 24, 1986Dec 29, 1987Plant Genetics, Inc.Artificial seed coat for botanic seed analogsUS4769945 *Jan 30, 1987Sep 13, 1988Kirin Brewery Co., Ltd.Delivery unit of plant tissueUS4777762 *Dec 24, 1986Oct 18, 1988Plant Genetics, Inc.Process for creating an analog to natural botanic seedUS4779376 *Apr 14, 1986Oct 25, 1988Plant Genetics, Inc.Encapsulation seed and adjuvantUS4780987 *Sep 9, 1985Nov 1, 1988Plant Genetics, Inc.Method for the preparation of hydrated, pregerminated seeds in gel capsulesUS4802305 *May 27, 1986Feb 7, 1989Sumitiomo Chemical Company, LimitedCoated seedsUS4806357 *Nov 25, 1987Feb 21, 1989The Regents Of The University Of CaliforniaVertical nozzle with dual vertical, rectilinear, concentric tubesUS4808430 *Feb 11, 1988Feb 28, 1989Yazaki CorporationEncapsulated with air bubbleUS4866096 *Mar 20, 1987Sep 12, 1989Air Products And Chemicals, Inc.Stable fluorochemical aqueous emulsionsUS4879839 *Apr 4, 1988Nov 14, 1989Solvay & Cie. (Societe Anonyme)Coated seeds and process for preparing themUS5010685 *May 1, 1989Apr 30, 1991Kirin Beer Kabushiki KaishaEncapsulated in mixture of ethylene-vinyl acetate copolymer and wax matricesUS5044116 *Dec 7, 1984Sep 3, 1991Interox (Societe Anonyme)Coated seeds and a process for their obtainmentUS5236469 *Oct 26, 1990Aug 17, 1993Weyerhaeuser CompanyOxygenated analogs of botanic seedCA1241552A1 *Oct 25, 1984Sep 6, 1988Charles E. NelsenCapsule production using biologically active substratesCA1250296A1 *Dec 29, 1982Feb 21, 1989The Green Cross CorporationPerfluorobicyclo compoundsEP0107141A1 *Oct 11, 1983May 2, 1984Plant Genetics, Inc.Method of vegetally propagating plants and analog to natural botanic seed for implementing said methodEP0380692A1 *Jul 25, 1989Aug 8, 1990Pentel Kabushiki KaishaPlant tissue culture processJPH0246240A * Title not availableJPS6140708A * Title not availableJPS62275604A * Title not availableJPS63133904A * Title not availableJPS63152905A * Title not availableWO1991001803A1 *Aug 8, 1990Feb 21, 1991Agronomique Inst Nat RechMultilayer granules containing coated active substances, production method, device for implementing such method and utilization of the granules obtained* Cited by examinerNon-Patent CitationsReference1"FLOUROMERT� Electronic Liquids for Direct Contact Dielectric Cooling" brochure, 3M Industrial Chemical Products Division, St. Paul, Minn. (1986).2"FLUORINERT� Electronic Liquids" brochure, 3M Industrial Chemical Products Division, St. Paul, Minn. (1989).3 *A. King et al. Bio/Technology, vol. 7 (1989) pp. 1037 1042.4A. King et al. Bio/Technology, vol. 7 (1989) pp. 1037-1042.5Adlercreutz and Mattiasson, "Oxygen Supply to Immobilized Biocatalysts. A Model Study," Acta Chem. Scand. B36:651-653 (1982).6Adlercreutz and Mattiasson, "Oxygen Supply to Immobilized Cells. 3. Oxygen Supply by Hemoglobin or Emulsions of Perfluorochemicals," Eur. J. Appl. Microbiol. & Biotechnol. 16:165-170 (1982).7Adlercreutz and Mattiasson, "Oxygen Supply to Immobilized Cells: 1. Oxygen Production by Immobilized Chlorella pyrenoidosa," Enzyme Microbiol. Technol. 4:332-336 (1982).8 *Adlercreutz and Mattiasson, Oxygen Supply to Immobilized Biocatalysts. A Model Study, Acta Chem. Scand. B36:651 653 (1982).9 *Adlercreutz and Mattiasson, Oxygen Supply to Immobilized Cells. 3. Oxygen Supply by Hemoglobin or Emulsions of Perfluorochemicals, Eur. J. Appl. Microbiol. & Biotechnol. 16:165 170 (1982).10 *Adlercreutz and Mattiasson, Oxygen Supply to Immobilized Cells: 1. Oxygen Production by Immobilized Chlorella pyrenoidosa, Enzyme Microbiol. Technol. 4:332 336 (1982).11Bapat and Rao, "Sandalwood Plantlets from `Synthetic Seeds,`" Plant Cell Reports 7:434-436 (1988).12 *Bapat and Rao, Sandalwood Plantlets from Synthetic Seeds, Plant Cell Reports 7:434 436 (1988).13Bapat et al., "In Vivo Growth of Encapsulated Axillary Buds of Mulberry, (Morus indica L.)," Plant Cell, Tissue and Organ Culture 20:69-70 (1990).14 *Bapat et al., In Vivo Growth of Encapsulated Axillary Buds of Mulberry, ( Morus indica L.), Plant Cell, Tissue and Organ Culture 20:69 70 (1990).15Bapat, "Studies on Synthetic Seeds of Sandalwood (Santalum album L. ) and Mulberry (Morus indica L.)," in Synseeds: Applications of Synthetic Seeds to Crop Improvement, Redenbaugh, Ed., CRC Press, Florida (1993), chap. 21.16 *Bapat, Studies on Synthetic Seeds of Sandalwood ( Santalum album L. ) and Mulberry ( Morus indica L.), in Synseeds: Applications of Synthetic Seeds to Crop Improvement, Redenbaugh, Ed., CRC Press, Florida (1993), chap. 21.17Chandler et al., "Effects of Emulsified Perfluorochemicals on Growth and Ultrastructure of Microbial Cells in Culture," Biotechnol. Letters 9:195-200 (1987).18 *Chandler et al., Effects of Emulsified Perfluorochemicals on Growth and Ultrastructure of Microbial Cells in Culture, Biotechnol. Letters 9:195 200 (1987).19Clark et al., "Emulsions of Perfluorinated Solvents for Intravascular Gas Transport," Fed. Proceed. 34:1468-1477 (1975).20Clark et al., "The Physiology of Synthetic Blood," J. Thorac. & Cardiovasc. Surg. 60:757-773 (1970).21 *Clark et al., Emulsions of Perfluorinated Solvents for Intravascular Gas Transport, Fed. Proceed. 34:1468 1477 (1975).22 *Clark et al., The Physiology of Synthetic Blood, J. Thorac. & Cardiovasc. Surg. 60:757 773 (1970).23Damiano and Wang, "Novel Use of a Perfluorocarbon for Supplying Oxygen to Aerobic Submerged Cultures," Biotechnol. Letters 7:81-86 (1985).24 *Damiano and Wang, Novel Use of a Perfluorocarbon for Supplying Oxygen to Aerobic Submerged Cultures, Biotechnol. Letters 7:81 86 (1985).25Datta and Potrykus, "Artificial Seeds in Barley: Encapsulation of Microspore-Derived Embryos," Theor. Appl. Genet. 77:820-824 (1989).26 *Datta and Potrykus, Artificial Seeds in Barley: Encapsulation of Microspore Derived Embryos, Theor. Appl. Genet. 77:820 824 (1989).27Davis et al., "Novel Compositions of Emulsified Perfluorocarbons for Biological Applications," Brit. J. Pharmacol. 89:665P (1986).28 *Davis et al., Novel Compositions of Emulsified Perfluorocarbons for Biological Applications, Brit. J. Pharmacol. 89:665P (1986).29 *FLOUROMERT Electronic Liquids for Direct Contact Dielectric Cooling brochure, 3M Industrial Chemical Products Division, St. Paul, Minn. (1986).30 *FLUORINERT Electronic Liquids brochure, 3M Industrial Chemical Products Division, St. Paul, Minn. (1989).31Fujii et al., "ABA Maturtion and Starch Accumulation in Alfalfa Somatic Embryos" (Abstract), In Vitro 25 (No. 3, Part 2): 61A (1989).32Fujii et al., "Improving Plantlet Growth and Vigor from Alfalfa Artificial Seed" (Abstract), in Vitro 24 (No. 3, Part 2):70A (1989).33 *Fujii et al., ABA Maturtion and Starch Accumulation in Alfalfa Somatic Embryos (Abstract), In Vitro 25 (No. 3, Part 2): 61A (1989).34 *Fujii et al., Improving Plantlet Growth and Vigor from Alfalfa Artificial Seed (Abstract), in Vitro 24 (No. 3, Part 2):70A (1989).35Fujii et et al., "Artificial Seeds for Plant Propagation," Trends in Bio/Technol. 5:335-339 (1987).36 *Fujii et et al., Artificial Seeds for Plant Propagation, Trends in Bio/Technol. 5:335 339 (1987).37Fujita et al., "Fluorocarbon Emulsion as a Candidate for Artificial Blood," Europ, Surg. Res. 3:436-453 (1971).38 *Fujita et al., Fluorocarbon Emulsion as a Candidate for Artificial Blood, Europ, Surg. Res. 3:436 453 (1971).39Geyer, R. P., "`Bloodless` Rats Through the Use of Artificial Blood Substitutes," Fed. Proceed. 34:1499-1505 (1975).40 *Geyer, R. P., Bloodless Rats Through the Use of Artificial Blood Substitutes, Fed. Proceed. 34:1499 1505 (1975).41Gupta and Durzan, "Biotechnology of Somatic Polyembryogenesis and Plantlet Regeneration in Loblolly Pine," Bio/Technol. 5:147-151 (1987).42 *Gupta and Durzan, Biotechnology of Somatic Polyembryogenesis and Plantlet Regeneration in Loblolly Pine, Bio/Technol. 5:147 151 (1987).43Ibarbia, "Synthetic Seed: Is it the Future," Western Grower and Shipper 59:12 (1988).44 *Ibarbia, Synthetic Seed: Is it the Future, Western Grower and Shipper 59:12 (1988).45Janick, "Production of Synthetic Seed via Desiccation and Encapsulation" (Abstract), In Vitro 24 (No. 3, Part 2):70A (1989).46 *Janick, Production of Synthetic Seed via Desiccation and Encapsulation (Abstract), In Vitro 24 (No. 3, Part 2):70A (1989).47Kamada et al., "New Methods for Somatic Embryo Induction and Their Use for Synthetic Seed Production" (Abstract), In Vitro 24 (No. 3, Part 2):71A (1988).48 *Kamada et al., New Methods for Somatic Embryo Induction and Their Use for Synthetic Seed Production (Abstract), In Vitro 24 (No. 3, Part 2):71A (1988).49Kim and Janick, "ABA and Polyox-Encapsulation or High Humidity Increases Survival of Desiccated Somatic Embryos of Celery," Hortscience 24:674-676 (1989).50 *Kim and Janick, ABA and Polyox Encapsulation or High Humidity Increases Survival of Desiccated Somatic Embryos of Celery, Hortscience 24:674 676 (1989).51King et al., "Perfluorochemicals and Cell Culture," Biotechnol. 7:1037-1042 (1989).52 *King et al., Perfluorochemicals and Cell Culture, Biotechnol. 7:1037 1042 (1989).53Kitto and Janick, "A Citrus Embryo Assay to Screen Water-Soluble Resins as Synthetic Seed Coats," HortScience 20:98-100 (1985).54Kitto and Janick, "Production of Synthetic Seeds by Encapsulating Asexual Embryos of Carrot," J. Amer. Soc. Hort. Sci. 110:277-282 (1985).55 *Kitto and Janick, A Citrus Embryo Assay to Screen Water Soluble Resins as Synthetic Seed Coats, HortScience 20:98 100 (1985).56 *Kitto and Janick, Production of Synthetic Seeds by Encapsulating Asexual Embryos of Carrot, J. Amer. Soc. Hort. Sci. 110:277 282 (1985).57Li, "Somatic Embryogenesis and Synthetic Seed Technology Using Carrot as a Model System," in Synseeds: Applications of Synthetic Seeds to Crop Improvement, Redenbaugh, Ed., CRC Press, Florida (1993), chap. 16.58 *Li, Somatic Embryogenesis and Synthetic Seed Technology Using Carrot as a Model System, in Synseeds: Applications of Synthetic Seeds to Crop Improvement, Redenbaugh, Ed., CRC Press, Florida (1993), chap. 16.59Mattiason and Adlercreutz, "Use of Perfluorochemicals for Oxygen Supply to Immobilized Cells," Ann. N.Y. Acad. Sci. 413:545-547 (1984).60 *Mattiason and Adlercreutz, Use of Perfluorochemicals for Oxygen Supply to Immobilized Cells, Ann. N.Y. Acad. Sci. 413:545 547 (1984).61Redenbaugh et al., "Encapsulated Plant Embryos," Advances in Biotechnical Processes 9:225-248 (1988).62Redenbaugh et al., "Encapsulation of Somatic Embryos in Synthetic Seed Coats," HortScience 21 (No. 3, Section 2): 819-820 (1986) (Abstract of presentation at XXII Int'l Hortic. Cong., Aug. 10-18, 1986, Davis, Calif.).63Redenbaugh et al., "Encapsulation of Somatic Embryos in Synthetic Seed Coats," HortScience 22:803-809 (1987).64Redenbaugh et al., "III.3 Artificial Seeds-Encapsulated Somatic Embryos," Biotech. in Agr. & For. 17:395-416 (1991).65Redenbaugh et al., "Scale-Up: Artificial Seeds," In Green et al. (eds.), Plant Tissue and Cell Culture, pp. 473-493, Alan R. Liss, NY (1987).66Redenbaugh et al., "Somatic Seeds: Encapsulation of Asxeual Plant Embryos," Bio/Technol. 4:797-801 (1986).67 *Redenbaugh et al., Encapsulated Plant Embryos, Advances in Biotechnical Processes 9:225 248 (1988).68 *Redenbaugh et al., Encapsulation of Somatic Embryos in Synthetic Seed Coats, HortScience 21 (No. 3, Section 2): 819 820 (1986) (Abstract of presentation at XXII Int l Hortic. Cong., Aug. 10 18, 1986, Davis, Calif.).69 *Redenbaugh et al., Encapsulation of Somatic Embryos in Synthetic Seed Coats, HortScience 22:803 809 (1987).70 *Redenbaugh et al., III.3 Artificial Seeds Encapsulated Somatic Embryos, Biotech. in Agr. & For. 17:395 416 (1991).71 *Redenbaugh et al., Scale Up: Artificial Seeds, In Green et al. (eds.), Plant Tissue and Cell Culture, pp. 473 493, Alan R. Liss, NY (1987).72 *Redenbaugh et al., Somatic Seeds: Encapsulation of Asxeual Plant Embryos, Bio/Technol. 4:797 801 (1986).73Redengaugh et al., "Encapsulation of Somatic Embryos for Artificial Seed Production" (Abstract), In Vitro 20 (Part 2): 256-257 (1984).74 *Redengaugh et al., Encapsulation of Somatic Embryos for Artificial Seed Production (Abstract), In Vitro 20 (Part 2): 256 257 (1984).75Riess and Le Blanc, "Perfluoro Compounds as Blood Substitutes," Angew. Chem. Int. Ed. Engl. 17:621-634 (1978).76 *Riess and Le Blanc, Perfluoro Compounds as Blood Substitutes, Angew. Chem. Int. Ed. Engl. 17:621 634 (1978).77Rogers, "Synthetic-Seed Technology," Newsweek, Nov. 28, 1983.78 *Rogers, Synthetic Seed Technology, Newsweek, Nov. 28, 1983.79Sanada et al., "Celery and Lettuce," in Synseeds: Applications of Synthetic Seeds to Corp Inprovement, Redenbaugh, Ed., CRC Press, Florida (1993), Chap. 17.80 *Sanada et al., Celery and Lettuce, in Synseeds: Applications of Synthetic Seeds to Corp Inprovement, Redenbaugh, Ed., CRC Press, Florida (1993), Chap. 17.81Senaratna, "Artificial Seeds," Biotech. Adv. 10:379-392 (1992).82 *Senaratna, Artificial Seeds, Biotech. Adv. 10:379 392 (1992).83Stuart and Redenbaugh, "Use of Somatic Embryogenesis for the Regeneration of Plants,"in LaBaron et al. (eds.), Biotechnology in Agricultural Chemistry, Ch. 6, pp. 87-96, American Chemical Society, Washington, D.C. (1987).84 *Stuart and Redenbaugh, Use of Somatic Embryogenesis for the Regeneration of Plants, in LaBaron et al. (eds.), Biotechnology in Agricultural Chemistry, Ch. 6, pp. 87 96, American Chemical Society, Washington, D.C. (1987).85Teasdale and Buxton, "Culture of Pinus Radiat Embryos with Reference to Artificial Seed Production," New Zealand J. For. Sci. 16:387-391 (1986).86 *Teasdale and Buxton, Culture of Pinus Radiat Embryos with Reference to Artificial Seed Production, New Zealand J. For. Sci. 16:387 391 (1986).* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS6340594Dec 5, 1997Jan 22, 2002Cellfor, Inc.Production of desiccation-tolerant gymnosperm embryosUS6372496Dec 5, 1997Apr 16, 2002Cellfor, Inc.Mature viable gymnosperm somatic embryoUS6444467Jun 11, 1999Sep 3, 2002Cellfor Inc.Pre-germinating somatic 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, and propagating the sownUS6689609Apr 14, 2000Feb 10, 2004Cellfor Inc.Enhancing germination of plant somatic embryos by primingUS6946295 *Jun 11, 1999Sep 20, 2005Cellfor, Inc.Process for ex vitro sowing and germination of plant somatic embryosUS7795029Dec 4, 2003Sep 14, 2010Cellfor Inc.Using nutrient broth and porous solid support to culture and sow heterotrophic somatic plant embryo or germinant of conifer; tissue engineering and plant propagationUS7882656Jun 9, 2009Feb 8, 2011Weyerhaeuser Nr CompanyManufactured seed having an improved end sealUS7923249Mar 10, 2006Apr 12, 2011Cellfor Inc.Aerated liquid priming of conifer somatic germinantsWO1999065293A1Jun 11, 1999Dec 23, 1999Potter Ann Kathryn EastmanA process for production and subsequent ex vitro sowing and propagation of pre-germinated plant somatic embryos* Cited by examinerClassifications U.S. Classification47/57.6, 435/1.1, 47/58.10R, 435/430.1International ClassificationA01H4/00, A01C1/00, A01C1/06Cooperative ClassificationY10S47/09, Y10S47/11, A01H4/006European ClassificationA01H4/00D1Legal EventsDateCodeEventDescriptionApr 21, 2009ASAssignmentOwner name: WEYERHAEUSER NR COMPANY, WASHINGTONFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:WEYERHAEUSER COMPANY;REEL/FRAME:022835/0233Effective date: 20090421Owner name: WEYERHAEUSER NR COMPANY,WASHINGTONFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:WEYERHAEUSER COMPANY;REEL/FRAME:22835/233Jun 21, 2007FPAYFee paymentYear of fee payment: 12Jun 24, 2003FPAYFee paymentYear of fee payment: 8Jun 23, 1999FPAYFee paymentYear of fee payment: 4Aug 20, 1996CCCertificate of correctionRotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google