Weyerhaeuser Company Limited

Weyerhaeuser Nr Company

Other

University of California

Pramod K Gupta

Bonnie Larson

Diane Gail Holmstrom

Gerald S Pullman

Roger Timmis

James A Grob

Don J Durzan

Carolyn V Carpenter

Martha K Koester

Pamela L Carlson

Doris Budworth

Susan D Rayfield

Judith Zucati

Methods of initiating plant somatic embryos from megagametophytes are provided.

Methods of initiating plant somatic embryos

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).

Methods of multiplying conifer embryogenic tissue

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.

Methods and compositions for regrowth of cryopreserved conifer embryos

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.

Methods for increasing germination vigor by early singulation of conifer somatic embryos

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.

Methods for producing a synchronized population of conifer somatic embryos

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.

Development and stratification of pine somatic embryos using a liquid system

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.

Continuous culture of conifer embryogenic tissue

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.

Methods for producing high yields of zygotic-like cotyledonary pine embryos utilizing media that include a disaccharide and glucose

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.

Methods for promoting maturation of conifer somatic embryos

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.

Methods for developing conifer somatic embryos

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.

Methods for producing cotyledonary pine embryos utilizing a gibberellin

The invention concerns measurement of sucrose series oligosaccharides, particularly sucrose, raffinose, and stachyose, and the dehydrin group proteins, in conifer somatic embryos as a measure of their biochemical maturity and readiness to germinate. The information gained is useful for evaluation of the effectiveness of the culture media, particularly that used for the development of the cotyledonary embryo stage. Somatic embryos having elevated levels of the oligosaccharides have been shown to have improved germination, especially those in the genus Pinus. The invention is also directed to conifer somatic embryos having elevated levels of sucrose series oligosaccharides and dehydrin group proteins.

Method for determining maturity of conifer somatic embryos

A method for clonal propagation by somatic polyembryogenesis. The method allows for clonal propagation of embryonal suspensor mass resulting in true-to-type suspensor development of the conifer embryo leading to development of plantlets and plants.

Method for clonal propagation of gymnosperms by somatic polyembryogenesis

The invention is a method for reproducing coniferous trees by somatic embryogenesis using plant tissue culture techniques in a multistage culturing process. A suitable explant, typically the fertilized embryo excised from an immature seed, is first cultured on a medium that induces multiple early stage proembryos. These are multiplied in a second culture having reduced growth hormones. Maltose is supplied as the carbon and energy source in the second culture. Alternatively, a sucrose-containing maintenance and multiplication medium may be used followed by at least two subcultures on a maltose based maintenance medium. The early stage embryos grow in size and vigor to advanced early stage embryos. The embryos are then transferred to a cotyledonary embryo development culture. After several weeks somatic embryos having the appearance of zygotic embryos will have formed. These may be germinated before or after storage and transplanted to soil for further growth. Maltose used in the maintenance and multiplication culture results in larger and more robust advanced early stage embryos which, in turn, produce cotyledonary embryos very similar in morphology to natural zygotic embryos. The use of maltose at earlier stages of embryo development is more important than its use for embryo maturation.

Method for reproducing conifers by somatic embryogenesis using a maltose

The invention is a method for reproducing Douglas-fir by somatic embryogenesis using plant tissue culture techniques in a multistage culturing process. A suitable explant, typically the fertilized embryo excised from an immature seed, is first cultured on a medium that induces multiple early stage embryos. These are multiplied in a second culture having reduced growth hormones where the early stage embryos grow in size and vigor to advanced early stage embryos. The embryos may then be transferred directly to a cotyledonary embryo development culture containing an adsorbent such as activated charcoal. Preferably, they are first singulated by shaking in two or more liquid subcultures containing the adsorbent prior to placing them on the development medium. After several weeks somatic embryos having the appearance of zygotic embryos will have formed. These may be germinated before or after storage and transplanted to soil for further growth.

Growing community of inventors

Federal Way, WA, United States of America

Pramod K Gupta