Source: http://www.google.com/patents/US5666762?dq=U.S.+Patent+No.+4,528,643
Timestamp: 2016-08-28 16:42:15
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Patent US5666762 - Respiration-limited manufactured seed - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsThe present invention provides manufactured seeds comprising totipotent plant tissue and a hydrated gel, preferably surrounded by a protective seed coat. The cotyledons of the germinating totipotent plant tissue can be enclosed in a shoot restraint that is resistant to penetration by the growing cotyledons....http://www.google.com/patents/US5666762?utm_source=gb-gplus-sharePatent US5666762 - Respiration-limited manufactured seedAdvanced Patent SearchPublication numberUS5666762 APublication typeGrantApplication numberUS 08/487,600Publication dateSep 16, 1997Filing dateJun 7, 1995Priority dateOct 26, 1990Fee statusPaidAlso published asCA2094511A1, CA2094511C, CA2161814A1, CA2161814C, DE69131052D1, EP0607140A1, EP0607140A4, EP0607140B1, US5427593, US5564224, US5687504, US5701699, US5732505, WO1992007457A1Publication number08487600, 487600, US 5666762 A, US 5666762A, US-A-5666762, US5666762 A, US5666762AInventorsWilliam C. Carlson, Michael K. McKinnis, Jeffrey E. HartleOriginal AssigneeWeyerhaeuser CompanyExport CitationBiBTeX, EndNote, RefManPatent Citations (38), Non-Patent Citations (100), Referenced by (34), Classifications (11), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetRespiration-limited manufactured seed
US 5666762 AAbstract
The present invention provides manufactured seeds comprising totipotent plant tissue and a hydrated gel, preferably surrounded by a protective seed coat. The cotyledons of the germinating totipotent plant tissue can be enclosed in a shoot restraint that is resistant to penetration by the growing cotyledons. The manufactured seeds can be prepared for long-term storage, e.g., by sparging the hydrated gel with a respiration-limiting gas before, during, or after assembly of the manufactured seed. The seed is then preferably stored in an atmosphere containing levels of a respiration-limiting gas sufficient to keep respiration of the totipotent plant tissue at reduced levels.
1. A manufactured seed comprising a unit of totipotent plant tissue and a hydrated gel disposed relative to the plant tissue so as to allow liquid transfer from the gel to the plant tissue, the hydrated gel comprising a concentration of a respiration-limiting gas effective to reduce respiration of the plant tissue to a level less than respiration of totipotent plant tissue in a control manufactured seed.
2. The manufactured seed of claim 1 wherein the gas is selected from the group consisting of carbon dioxide and nitrogen.
3. The manufactured seed of claim 1 wherein the gel has an free oxygen concentration below about 10%.
4. The manufactured seed of claim 1 wherein the gel further comprises an inert oxygen-carrying compound.
5. The manufactured seed of claim 4 wherein the oxygen-carrying compound is a member of the group consisting of a perfluorocarbon and a silicone oil.
6. The manufactured seed of claim 1 further comprising a manufactured seed coat at least partially surrounding the gel and the plant tissue.
7. The manufactured seed of claim 6 wherein the seed coat comprises a gas-impermeable shell and defines an opening in the shell.
8. The manufactured seed of claim 7 wherein a gas-permeable end seal is disposed across the opening.
9. The manufactured seed of claim 7 wherein the totipotent plant tissue is sufficiently developed to comprise a radicle and the radicle is oriented toward the opening.
10. The manufactured seed of claim 1 wherein the totipotent plant tissue is sufficiently developed to comprise a shoot end, the manufactured seed further comprising a restraint enclosing at least a portion of the shoot end.
11. A method of storing a manufactured seed comprising a unit of totipotent plant tissue and a hydrated gel disposed relative to the plant tissue so as to allow liquid transfer from the gel to the plant tissue, the method comprising the steps of:(a) exposing the manufactured seed to an atmosphere comprising a first respiration-limiting gas for a time sufficient for the gel have a concentration of the respiration-limiting gas sufficient to substantially reduce respiration of the plant tissue; then (b) storing the manufactured seed in the presence of a concentration of a second respiration-limiting gas sufficient to substantially reduce respiration of the plant tissue, thereby producing a stored seed. 12. The method of claim 11 wherein the first or second gas is selected from the group consisting of carbon dioxide and nitrogen.
13. The method of claim 11 wherein the first gas is the same as the second gas.
14. The method of claim 11 further comprising step (c) of exposing the stored seed to an atmosphere comprising molecular oxygen until the gel provides a gas environment that supports germination of the plant tissue.
15. A method of producing a manufactured seed comprising the steps of:(a) providing a unit of totipotent plant tissue; (b) providing a hydrated gel; (c) disposing the plant tissue relative to the hydrated gel so as to allow liquid transfer from the gel to the plant tissue, thereby producing a manufactured seed; and (d) exposing the hydrated gel to an atmosphere comprising a respiration-limiting gas for a time sufficient for the gel have a concentration of the respiration-limiting gas sufficient to substantially reduce respiration of the plant tissue. 16. The method of claim 15 wherein step (d) is performed after step (c).
17. A method of producing a manufactured seed comprising the steps of:(a) providing a unit of totipotent plant tissue; (b) disposing the plant tissue relative to a hydrated gel so as to allow liquid transfer from the gel to the plant tissue, the gel comprising a concentration of a respiration-limiting gas sufficient to substantially reduce respiration of the plant tissue, thereby producing the manufactured seed. 18. The method of claim 17 further comprising step (c) of exposing the seed to a gas environment comprising a level of molecular oxygen effective to increase respiration of the plant tissue to a level permitting germination of the plant tissue.
In view of the disadvantages of producing large numbers of identical progeny plants by sexual means, propagation of commercially valuable plants via culturing of somatic or zygotic plant embryos has been intensively studied. Such "asexual" propagation has been shown for some species to yield large numbers of genetically identical embryos each having the capacity to develop into a normal plant. Unfortunately, these embryos, which are produced under laboratory conditions, lack the protective and nutritive structures found in botanic seeds. As a result, the embryos must usually be further cultured under laboratory conditions until they reach an autotrophic "seedling" state characterized by an ability to produce their own food via photosynthesis, resist desiccation, produce roots able to penetrate soil, and fend off soil microorganisms. Such extensive laboratory culture during several distinct stages in plant development is time-consuming, resource-intensive, and requires skilled labor.
The hydrated gels used for producing manufactured seeds rapidly lose water to the ambient air or soil on sowing and fail to provide optimal protection to the embryo from mechanical damage resulting from handling and mechanical sowing and from attack by various plant pathogens, herbivores, or other pests, either before or after germination.
In agriculture, natural seed is normally sown during a relatively short period of the biological year. In order to plant large quantities of manufactured seed in this relatively short sowing period, either extremely rapid manufacturing procedures must be developed or the seeds must be stored and thereby accumulated as they are manufactured. It would be possible to develop equipment for manufacturing seed at a speed that would support sowing large crops without long-term seed storage, but seed storage would make the production of manufactured seed more efficient. It is therefore desirable to develop methods that would allow accumulation and long-term storage of manufactured seed. It is also desirable to develop manufactured seeds capable of extended storage without substantial loss of viability before sowing. The present invention meets these and other needs.
The present invention provides manufactured seeds comprising a plant embryo or other unit of totipotent plant tissue. The totipotent plant tissue is preferably protected from mechanical damage, desiccation, and attack by pathogens, pests, etc. by a manufactured seed coat. In one embodiment of the present invention, the seed coat comprises a gas-impermeable shell defining an opening, across which is preferably disposed a gas-permeable seal.
The totipotent plant tissue is preferably disposed relative to, and preferably surrounded by or in contact with, a hydrated gel so as to allow the transfer of liquid, dissolved solutes, and gases from the gel to the plant tissue. The gel can comprise an inert oxygen-absorbing or oxygen-carrying compound such as a perfluorocarbon or silicone oil. The gel serves as a "synthetic gametophyte" for the totipotent plant tissue in a manner analogous to the gametophyte portion of a natural botanic seed, i.e., the endosperm or other seed nutritive tissue, depending upon the species from which the totipotent plant tissue originates.
Such a manufactured seed can be prepared for long-term storage, for example, if the hydrated gel comprises an amount of a respiration-limiting gas sufficient to substantially reduce respiration of the totipotent plant tissue, including, for example, carbon dioxide or nitrogen. This can be accomplished, for example, by exposing the gel to the respiration-limiting gas before, during, or after assembly of the manufactured seed, i.e., before the totipotent plant tissue is inserted into or otherwise disposed in fluid contact with the gel. It is preferable, after exposure of the gel to the respiration-limiting gas and/or assembly of the manufactured seed to store the manufactured seed in the presence of a concentration of the same or a different respiration-limiting gas to keep respiration of the totipotent plant tissue at reduced levels. Before sowing the manufactured seed, the stored seed can simply be exposed to an oxygen-containing atmosphere such as air or an oxygen-enriched atmosphere so as to provide the totipotent plant tissue with a gas environment that supports germination of the plant tissue, preferably until levels of the respiration-limiting gas inside the seeds drop, and/or levels of molecular oxygen inside the seeds rise, to a level that supports germination.
A number of appropriate nutrient formulations exist in the art, including a number of proprietary formulations. For example, a popular medium is the "MS liquid" (Murashige and Skoog, Physiologia Plantatum 15:473-497 (1962)) containing the following dissolved in water:
______________________________________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/L Myoinositol       100     mgIAA                10      mg/LSucrose            30000   mg/LpH                 5.7-5.8______________________________________
As used herein, a "nutrient liquid" is an aqueous solution of nutrients similar to the "MS liquid" formulation. A "nutrient agar" s similar to the "MS medium." Changes in types and amounts of certain ingredients can be made to meet the needs of specific types of plants without departing in any substantial manner from the purpose and utility of a nutrient liquid or nutrient medium.
Various plant-growth regulators, alone or in combination can also be added to the artificial gametophyte or one or more seed coats of the manufactured seed. Representative plant-growth regulators include auxins, cytokinins, gibberellins (e.g., GA3, GA4n, etc.), or other plant-growth regulators known in the art. It is preferred that such plant-growth regulators be present in the artificial gametophyte.
The FIG.-3C embodiment is similar to the FIG.-3B embodiment with respect to the bullet shape of the seed 90. The seed 90 comprises a nutrient unit 94, and a seed coat 95 having a tapered first end 93 which terminates with an orifice 98 and second end 96. A hydrated gel block 91 in which the totipotent plant tissue 12 is embedded is shown as an ovoid shape rather than the cylindrical shape of the gel block 81 in FIG. 3B. The FIG.-3C embodiment illustrates that the gel block 91 containing the totipotent plant tissue can be formed separately instead of being cast in the seed coat as suggested in FIG. 3B. The separate nutrient unit 94 can be eliminated by incorporating the nutrients into the gel comprising gel block 91.
"Radicle germination" denotes the emergence or protrusive growth of the-primary root from the capsule, caused by elongation of the radicle sufficient to burst the capsule. This term does not take into consideration any length criteria.
The following example is based on the manufactured seed design comprising a unit of totipotent plant tissue, seed coat, restraint, and hydrated gel as shown in FIG. 7. The manufactured seeds can be exposed to a respiration-limiting gas mixture having a reduced level of oxygen and an increased level of nitrogen (relative to atmospheric) for a time sufficient to equilibrate the concentration of oxygen and nitrogen in the gel with that in the gas mixture.
Manufactured seed (lacking totipotent plant tissue) can be tested by equilibrating the capsule with any of various test gas mixtures in the same fashion. The totipotent plant tissue is then inserted into the capsule just before sowing. In this case, the totipotent plant tissue is either (1) desiccated and stored separately in the respiration-limiting gas mixture or (2) fresh.
After infusing the manufactured seed with the respiration-limiting gas mixture, the seed are sealed in an airtight container the headspace of which is filled with the same respiration-limiting gas mixture. The seed are then be stored at 1� C. for one month. After storage, the seed are sown in soil and the rate of germination and percentage normal germinants determined.
The following gas mixtures are tested:
1. 0% oxygen, 100% nitrogen.
2. 2.5% oxygen, 97.5% nitrogen.
3. 5.0% oxygen, 95.0% nitrogen.
4. 7.5% oxygen, 92.5% nitrogen.
5. Air (i.e., atmospheric level of oxygen).
6. Control: seed prepared just prior to sowing.
7. Seed purged with 100% oxygen, then stored one month at 1� C. in air.
8. Seed purged with 100% oxygen, then stored one month at room temperature (about 20� C.) in air.
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