Abstract: Provided is a method of producing plant embryos having improved embryo quality or germination frequency comparing to embryos developed by conventional methods. The method entails the steps of (a) incubating plant embryogenic suspensor mass (ESM) in, or on, a standard development medium supplied with glucose for a first incubation period to develop immature somatic embryos; (b) mass transferring the immature somatic embryos to a modified development medium near or at cotyledon stage; and (c) incubating the immature somatic embryos in, or on, the modified development medium for a second incubation period to develop mature somatic embryos. The modified development medium contains reduced concentration of glucose or is glucose-free and the osmolality of the modified development medium is adjusted to compensate for the loss of osmolality due to reduction or removal of glucose.

Abstract: Methods of developing plant somatic embryos include incubating the plant somatic embryos at a first temperature for a first period of time followed by storing the plant somatic embryos at a second temperature for a second period of time. The first temperature can be in the range of about 23° C. to 30° C. and leads to rapid plant somatic embryo growth. The second temperature can be in the range of about 8° C. to 15° C. and slows plant somatic embryo development. Plant somatic embryos developed in this manner and implanted in artificial seeds show germination rates similar to zygotic embryos implanted in artificial seeds.

Abstract: A method of improving germination of plant embryos is provided. The method entails treating a culture of plant tissue with a liquid multiplication medium comprising a plant non-metabolizable sugar. Also provided is a multiplication medium for liquid cultures of plant cells comprising isomaltulose.

Abstract: A method for developing conifer cotyledonary somatic embryos is described. The method includes culturing conifer pre-cotyledonary somatic embryos in, or on, a development medium at a temperature from about 20° C. to about 22° C. for a first incubation period sufficient in length for at least a portion of the pre-cotyledonary somatic embryos to develop into cotyledonary somatic embryos, and culturing the cotyledonary somatic embryos in, or on, the development medium at a temperature from about 10° C. to about 15° C. for a second incubation period sufficient in length for at least a portion of the cotyledonary somatic embryos to reach anatomical maturity.

Abstract: Methods of initiating plant somatic embryos from megagametophytes are provided.

Abstract: The invention provides methods for multiplying conifer embryogenic tissue. The methods of the invention comprise the steps of: (a) culturing conifer embryogenic tissue in or on a solid multiplication medium comprising one or more growth hormones; and (b) culturing the conifer embryogenic tissue multiplied in step (a) in or on a liquid multiplication medium comprising one or more growth hormones, wherein the initial concentration of growth hormones in the solid multiplication medium of step (a) is greater than the initial concentration of growth hormones in the liquid multiplication medium of step (b).

Abstract: Methods of initiating plant somatic embryos from megagametophytes are provided.

Abstract: The invention provides methods for multiplying conifer embryogenic tissue. The methods of the invention comprise the steps of: (a) culturing conifer embryogenic tissue in or on a solid multiplication medium comprising one or more growth hormones; and (b) culturing the conifer embryogenic tissue multiplied in step (a) in or on a liquid multiplication medium comprising one or more growth hormones, wherein the initial concentration of growth hormones in the solid multiplication medium of step (a) is greater than the initial concentration of growth hormones in the liquid multiplication medium of step (b).

Abstract: The present invention provides methods for regrowth of conifer embryogenic cells. The methods comprise the steps of (a) contacting cryopreserved conifer embryogenic cells with a liquid transition medium, and (b) culturing the contacted conifer embryogenic cells in a regrowth medium to generate regrowth of the conifer embryogenic cells. Generally, the cryopreserved conifer embryogenic cells are contacted with the liquid transition medium for less than about 24 hours, such as for about one hour.

Abstract: In one aspect, a method is provided for increasing germination vigor of conifer somatic embryos produced in vitro. The method comprises (a) singulating a plurality of individual immature conifer somatic embryos from a first culture of embryos after a first incubation period in a first development media; and (b) contacting the plurality of singulated immature conifer somatic embryos with a second development media for a second incubation period.

Abstract: In one aspect, the present invention provides methods for producing conifer somatic embryos. The methods of this aspect of the invention each include the steps of cultivating conifer embryogenic cells in, or on, a synchronization medium that comprises an absorbent composition and at least one synchronization agent selected from the group consisting of abscisic acid and a gibberellin. Some embodiments of the methods provide a synchronized population of conifer somatic embryos wherein at least about 50% of the embryos are at about the same developmental stage. Some embodiments of the methods of the invention yield at least 100% more cotyledonary somatic embryos than an identical method for producing conifer somatic embryos that does not utilize a synchronization medium comprising an absorbent composition and at least one synchronization agent selected from the group consisting of abscisic acid and a gibberellin.

Abstract: The invention provides methods for producing pine somatic embryos using a liquid development medium and/or a liquid stratification medium. In a first aspect, the methods comprise the step of culturing embryogenic cells in, or on, a liquid development medium to produce cotyledonary pine somatic embryos. In another aspect, the methods comprise the step of culturing pine cotyledonary somatic embryos in, or on, a liquid stratification medium to produce stratified cotyledonary somatic embryos. The invention also provides methods for producing pine somatic embryos in bioreactors.

Abstract: The present invention provides methods for multiplying conifer embryogenic tissue. The methods of the present invention each include the step of continuously culturing conifer embryogenic tissue in liquid multiplication medium for a period of time sufficient for the embryogenic tissue to multiply.

Abstract: The present invention provides methods for producing cotyledonary pine embryos. The methods of the invention each include the step of culturing embryogenic pine tissue in, or on, a medium comprising a disaccharide and glucose to yield cotyledonary pine embryos, wherein the disaccharide and glucose are each present in the medium at a concentration of less than 3 percent. The present invention also provides cotyledonary pine embryos prepared by a method of the invention.

Abstract: In one aspect, the present invention provides methods for producing mature conifer somatic embryos. The methods of the invention comprise the steps of: (a) culturing pre-cotyledonary conifer somatic embryos in, or on, a development medium comprising less than 0.5% of gellan gum to form cotyledonary conifer somatic embryos; and (b) culturing cotyledonary conifer somatic embryos formed in step (a) in, or on, a maturation medium comprising between about 0.5% to about 1.5% of gellan gum to form mature conifer somatic embryos. In some embodiments, the maturation medium further comprises sucrose and/or abscisic acid.

Abstract: In one aspect, a method is provided for increasing germination vigor of conifer somatic embryos produced in vitro. The method comprises (a) singulating a plurality of individual immature conifer somatic embryos from a first culture of embryos after a first incubation period in a first development media; and (b) contacting the plurality of singulated immature conifer somatic embryos with a second development media for a second incubation period.

Abstract: The present invention provides methods for producing conifer cotyledonary somatic embryos. The methods of the present invention each includes the step of culturing embryogenic conifer tissue in, or on, a development medium for a period of time sufficient to produce conifer cotyledonary somatic embryos from the embryogenic conifer tissue, wherein the volume of development medium is in the range of from about 35 ml to about 50 ml per 0.5 mL of embryogenic conifer tissue.

Abstract: The present invention provides methods for producing cotyledonary pine embryos. The methods of the invention each include the step of culturing embryogenic pine tissue in, or on, a medium including at least one gibberellin to yield cotyledonary pine embryos. In some embodiments, an absorbent composition, such as activated charcoal, is included in the medium to further promote production of cotyledonary pine embryos.

Abstract: In one aspect, the present invention provides methods for producing Douglas-fir somatic embryos. In one aspect, the methods of the invention comprise the step of culturing Douglas-fir explants in, or on, an initiation medium comprising a principal carbohydrate source selected from the group consisting of maltose, glucose, and a combination thereof, to form embryonal suspensor masses comprising early-stage somatic embryos. In another aspect, the methods of the invention additionally comprise the step of culturing Douglas-fir embryonal suspensor masses in maintenance medium comprising a principal carbohydrate source selected from the group consisting of maltose, glucose, and a combination thereof, to form an established embryonal suspensor mass comprising early-stage somatic embryos.

Abstract: In one aspect, the present invention provides methods for producing conifer somatic embryos. The methods of this aspect of the invention each include the steps of cultivating conifer embryogenic cells in, or on, a synchronization medium that comprises an absorbent composition and at least one synchronization agent selected from the group consisting of abscisic acid and a gibberellin. Some embodiments of the methods provide a synchronized population of conifer somatic embryos wherein at least about 50% of the embryos are at about the same developmental stage. Some embodiments of the methods of the invention yield at least 100% more cotyledonary somatic embryos than an identical method for producing conifer somatic embryos that does not utilize a synchronization medium comprising an absorbent composition and at least one synchronization agent selected from the group consisting of abscisic acid and a gibberellin.