Weyerhaeuser Company Limited

Institute of Paper Science and Technology, Inc.

Other

Georgia Tech Research Corporation

Gerald S Pullman

Pramod K Gupta

Michael James Yancey

John Cairney

Ranjan Perera

Gary Peter

The present invention provides methods for initiating, capturing, maintaining and multiplying embryogenic cultures of coniferous plants. Methods include the use of novel media compositions containing Vitamin B, Vitamin E, or organic acids including &#x3b1;-ketoglutaric acid, pyruvic acid, or p-aminobenzoic acid to improve the frequency of embryogenic tissue initiation, capture, maintenance and multiplication. The methods are well suited for initiating embryogenic cultures in recalcitrant conifer varieties. The method is also well suited for producing somatic embryos that can be further cultured to produce large numbers of plants. Further, the invention provides novel methods that may be used to enhance somatic embryogenesis in a broad range of species.

Methods for improving conifer embryogenesis

The present invention provides methods for initiating embryogenic cultures of plants. The methods include the use of novel media compositions and elevated atmospheric pressure treatments to improve the frequency of embryogenic culture initiation. The methods are well suited for initiating embryogenic cultures in recalcitrant conifer varieties. The method is also well suited for producing somatic embryos that can be further cultured to produce large numbers of plants. Further, the invention provides novel methods that may be used to enhance somatic embryogenesis in a broad range of species.

Methods of initiating embryogenic cultures in plants

The present invention provides novel vicilin-like gene promoters. The promoter of the present invention may be operably linked to a desired sequence such as a gene or fragment thereof. A promoter-gene construct is also embodied by the present invention. Methods of producing and expressing polypeptides in plants are also provided. The present invention further provides methods for monitoring the embryo development of conifers.

Vicilin-like seed storage protein gene promoter and methods of using the same

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.

Method for reproducing douglas-fir by somatic embryogenesis

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. The early stage embryos may then be placed in or on a late stage proembryo development culture in order to develop very robust late stage proembryos having at least 100 cells. Culturing from this point continues in a cotyledonary embryo development medium containing an active gibberellin (GA) in an amount up to about 50 mg/L. Preferably exogenous abscisic acid (ABA) is also present in a similar amount. Concentration of GA and ABA may be reduced over time by inclusion of an adsorbent such as activated charcoal or by stepwise subcultures in which the later cultures have reduced hormone concentrations. 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. In addition to its use in the cotyledonary embryo development stage, GA may also be advantageously included in any of the maintenance cultures following embryo initiation. The use of GA results in larger and more robust somatic embryos and ultimately in greater germination success.

Method for reproducing conifers by somatic embryogenesis using mixed

The invention is a method for reproducing coniferous trees by somatic embryo-genesis 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. The early stage embryos may then be placed in or on a late stage proembryo development culture with a significantly higher osmotic potential than the previous stage or stages in order to develop very robust late stage proembryos having at least 100 cells. Culturing from this point continues in a cotyledonary embryo development medium having a raised osmotic potential. Exogenous abscisic acid is now required at this stage. The concentration of abscisic acid is decreased over time to low levels either by the use of an adsorbent such as activated charcoal or by stepwise subcultures in which the later cultures have lower ABA concentration. After a period of 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.

Method for reproducing conifers by somatic embryogenesis using stepwise

The invention is a method for treating a plant growth medium containing a plant so that the growth medium remains intact around the roots during transplanting operations. This involves treating the growth medium shortly before transplanting with an adhesive-forming substance which will bond the particles of the growth medium to form an intact plug. The adhesive-forming substance must be physiologically innocuous. Materials such as warmed solution of agar or soluble alginates have been found to be very suitable. When an alginate is used, it is rendered into an insoluble gel by secondary application of a chemical salt such as calcium nitrate. The method is particularly useful for growing seedlings or cuttings which must be transplanted while the root structure is still delicate and subject to damage from handling.

Method of transplanting a container grown plant

The invention lies in the field of conifer tissue culture. It is especially directed to a method for production of large concentrations of embryonal cells particularly suitable for genetic modification. The cells are enriched in the sense that the ratio of embryonal to nonembryogenic cells is very significantly in crased over that attained by earlier methods. The method comprises first developing an embryonalsuspensor mass containing early stage proembryos from a suitable explant. These are multiplied in a maintenance medium and developed to late stage proembryos in a medium that preferably has an osmotic level raised about 30-50% over the initiation medium. The late stage proembryos are transferred to a new medium that again has the osmotic level raised about 35-55% over the proembryo development medium. After three to six transfers a large number of embryonal cells without suspensors will have formed. These may be used in any of the known methods for genetic alteration.

High concentration enrichment of conifer embryonal cells

The invention is a method for developing tissue culture induced coniferous somatic proembryos into well developed cotyledonary embryos. The method comprises a multistage culturing process in which early stage proembryos are cultured on a late stage proembryo medium comprising a significantly higher osmotic potential along with abscisic acid and an absorbent material to gradually reduce the level of available abscisic acid over time. Culturing from this point continues in an embryo development medium having a high osmotic potential in which the osmotic potential is preferably raised during embryo development to a final level of about 450 mM/kg. Through this process the vigor and morphology of the embryos is improved and the tendency to germinate prematurely is significantly reduced. After a period of several weeks in culture somatic embryos having the appearance of zygotic embryos will have formed. These may be germinated before or after storage and transplanted to the soil for further growth.

Method for reproducing coniferous plants by somatic embryogenesis using

The invention is a method for reproducing coniferous trees by somatic embryogenesis using plant tissue culture techniques. It comprises a multistage culturing process. A suitable explant, typically the fertilized embryo excised from a mature or immature seed, is first cultured on a medium that induces multiple early stage proembryos. Preferably these proembryos are further multiplied in a second culture having reduced growth hormones. The early stage proembryos may then be placed in or on a late stage proembryo development culture which may have a significantly higher osmotic potential than the previous stage or stages to develop very robust late stage proembryos having at least about 100 cells and multiple suspensor cells. Culturing from this point continues in an embryo development medium very low in or lacking cytokinins and auxins but containing a relatively high concentration of exogenous abscisic acid and an adsorbent material, such as activated charcoal. The combination of abscisic acid and activated charcoal appears to increase the absolute number of embryos produced for most genotypes studied. Further, the vigor and morphology of the embryos is improved. After a period of several weeks cotyledonary embryos will have formed. These have a well defined bipolar structure with cotyledonary primordia at one end and a latent radicle at the other. If desired these embryos may be stored at about 4.degree. C. for extended periods while still on the development medium.

The present invention is a method for reproducing coniferous trees by somatic embryogenesis using plant tissue culture techniques. The method comprises a multistage culturing process. A suitable explant, typically the fertilized embryo excised from a mature or immature seed, is first cultured on a medium that induces multiple early stage proembryos. Preferably the proembryos from the indication stage are further multiplied in a second culture having reduced growth hormones. The early stage proembryos are then placed in or on a late stage proembryo development culture having a significantly higher osmotic potential than the previous stage or stages. This increased osmotic potential medium is a critical key to the development of very robust late stage proembryos having at least about 100 cells and multiple suspensor cells. Culturing from this point coninues in an embryo development medium very low in or lacking growth hormones but containing abscisic acid. After a period of several weeks cotyledonary embryos will have formed. These have a well defined bipolar structure with cotyledonary primordia at one end and a latent radicle at the other. Culturing to this point is carried out in darkness or greatly subdued light. The cotyledonary embryos are then transferred to a growth medium with a light/dark photoperiod for development of plantlets. The plantlets may then be transplanted to soil for further growth. The method has been successful with a broad range of species and with numerous genotypes that could not previously be propagated by embryogenesis.

Growing community of inventors

Renton, WA, United States of America

Gerald S Pullman