CA2274037A1 - Particle-mediated conifer transformation - Google Patents
Particle-mediated conifer transformation Download PDFInfo
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- CA2274037A1 CA2274037A1 CA002274037A CA2274037A CA2274037A1 CA 2274037 A1 CA2274037 A1 CA 2274037A1 CA 002274037 A CA002274037 A CA 002274037A CA 2274037 A CA2274037 A CA 2274037A CA 2274037 A1 CA2274037 A1 CA 2274037A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
- C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
Abstract
This invention relates to a method for genetically engineering coniferous plants. In particular, this invention relates to a particle-mediated gene transfer method for producing and developing transgenic somatic embryos for plants of the genus Pinus and Pinus interspecies hybrids. This method is well suited for producing transgenic clonal planting stock useful for reforestation.
Claims (73)
1. A method for genetically engineering conifers selected from the group consisting of the genus Pines and Pines interspecies hybrids, which comprises:
(a) placing conifer target tissue selected from the group consisting of embryogenic tissue containing pre-stage 3 somatic embryos, pre-stage 3 somatic embryos, pre-stage 3 zygotic embryos, and combinations thereof, on a target surface;
(b) bombarding the target tissue by physically accelerating at the target tissue carrier particles which are much smaller than the cells of the target tissue, the carrier particles carrying copies of a genetic construction including at least one gene of interest;
(c) inducing the bombarded target tissue to form proliferative tissue which is capable of forming somatic embryos;
(d) during the step of inducing, culturing the bombarded target tissue on selection medium so as to select for embryogenic tissue which is transformed by the gene of interest;
(e) inducing transformed somatic embryos to develop from the selected embryogenic tissue; and (f) germinating and converting the transformed somatic embryos thus produced into clonal transgenic conifer plants.
(a) placing conifer target tissue selected from the group consisting of embryogenic tissue containing pre-stage 3 somatic embryos, pre-stage 3 somatic embryos, pre-stage 3 zygotic embryos, and combinations thereof, on a target surface;
(b) bombarding the target tissue by physically accelerating at the target tissue carrier particles which are much smaller than the cells of the target tissue, the carrier particles carrying copies of a genetic construction including at least one gene of interest;
(c) inducing the bombarded target tissue to form proliferative tissue which is capable of forming somatic embryos;
(d) during the step of inducing, culturing the bombarded target tissue on selection medium so as to select for embryogenic tissue which is transformed by the gene of interest;
(e) inducing transformed somatic embryos to develop from the selected embryogenic tissue; and (f) germinating and converting the transformed somatic embryos thus produced into clonal transgenic conifer plants.
2. The method of claim 1 wherein the conifer is selected from the group consisting of Pinus taeda, Pinus serotina, Pinus palustris, Pinus elliottii, Pinus rigida, Pinus radiata, and hybrids thereof.
3. The method of claim 1 wherein the carrier particles are microparticles between 0.2 and 2.0 microns in diameter.
4. The method of claim 1 wherein the selection medium contains a sufficient amount of organic and inorganic nutrients, a selection agent at a concentration which is toxic to non-transformed cells but for which the gene of interest confers resistance to transformed cells, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 30.0 mg/l of abscisic acid, and up to about 60.0 g/l of sugar.
5. The method of claim 1 wherein the selection medium contains a sufficient amount of organic and inorganic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 30.0 mg/l of abscisic acid, up to about 60.0 g/l of sugar, and wherein the selection medium lacks a component necessary for the growth of non-transformed cells but for which the gene of interest confers to transformed cells the ability to produce the lacking component.
6. The method of claim 1 wherein the selection medium contains a sufficient amount of organic and inorganic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 30.0 mg/l of abscisic acid, up to about 60.0 g/l of sugar, and wherein the selection medium contains a component necessary for the growth of cells in a form which cannot be utilized by non-transformed cells but for which the gene of interest confers to transformed cells the ability to utilize the necessary component.
7. The method of claim 1 wherein the selection medium contains a sufficient amount of organic and inorganic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 30.0 mg/l of abscisic acid, up to about 60.0 g/l of sugar, and wherein the selection medium allows preferential growth of transformed cells containing the gene of interest.
8. The method of claims 4, 5, 6, and 7 wherein the sugar is a member selected from the group consisting of glucose, maltose, sucrose, and combinations thereof.
9. The method of claims 4, 5, 6, and 7 wherein the selection medium further contains a gelling agent selected from the group consisting of about 6.0 to about 9.0 g/l of agar, about 1.75 to about 4.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 5.0 g/l of AGARGEL, and combinations thereof.
10. The method of claim 1 which further comprises:
(a) culturing conifer target tissue selected from the group consisting of embryogenic tissue containing pre-stage 3 somatic embryos, pre-stage 3 somatic embryos, pre-stage 3 zygotic embryos, and combinations thereof, on preparation media containing a sufficient amount of inorganic and organic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 150.0 mg/l of abscisic acid, about 10.0 to about 120.0 g/l of sugar, and up to about 0.5M of organic alcohol, for a sufficient period of time to prepare the target tissue for bombardment by carrier particles;
(b) placing the prepared target tissue on a target surface;
(c) bombarding the prepared target tissue by physically accelerating at the prepared target tissue carrier particles which are much smaller than the cells of the target tissue, the carrier particles carrying copies of a genetic construction including at least one gene of interest;
(d) inducing the bombarded target tissue to form proliferative tissue which is capable of forming somatic embryos;
(e) during the step of inducing, culturing the bombarded target tissue on selection media so as to select for embryogenic tissue which is transformed by the gene of interest;
(f) inducing transformed somatic embryos to develop from the selected embryogenic tissue; and (g) germinating and converting the transformed somatic embryos thus produced into clonal transgenic conifer plants.
(a) culturing conifer target tissue selected from the group consisting of embryogenic tissue containing pre-stage 3 somatic embryos, pre-stage 3 somatic embryos, pre-stage 3 zygotic embryos, and combinations thereof, on preparation media containing a sufficient amount of inorganic and organic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 150.0 mg/l of abscisic acid, about 10.0 to about 120.0 g/l of sugar, and up to about 0.5M of organic alcohol, for a sufficient period of time to prepare the target tissue for bombardment by carrier particles;
(b) placing the prepared target tissue on a target surface;
(c) bombarding the prepared target tissue by physically accelerating at the prepared target tissue carrier particles which are much smaller than the cells of the target tissue, the carrier particles carrying copies of a genetic construction including at least one gene of interest;
(d) inducing the bombarded target tissue to form proliferative tissue which is capable of forming somatic embryos;
(e) during the step of inducing, culturing the bombarded target tissue on selection media so as to select for embryogenic tissue which is transformed by the gene of interest;
(f) inducing transformed somatic embryos to develop from the selected embryogenic tissue; and (g) germinating and converting the transformed somatic embryos thus produced into clonal transgenic conifer plants.
11. The method of claim 1 which further comprises (a) placing conifer target tissue selected from the group consisting of embryogenic tissue containing pre-stage 3 somatic embryos, pre-stage 3 somatic embryos, pre-stage 3 zygotic embryos, and combinations thereof, on a target surface;
(b) bombarding the target tissue by physically accelerating at the target tissue carrier particles which are much smaller than the cells of the target tissue, the carrier particles carrying copies of a genetic construction including at least one gene of interest;
(c) culturing the bombarded target tissue on preparation medium containing a sufficient amount of inorganic and organic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 150.0 mg/l of abscisic acid, about 10.0 to about 120.0 g/l of sugar, and up to about 0.5M of organic alcohol, for a sufficient period of time to allow the bombarded target tissue to recover from carrier particle insertion;
(d) inducing the bombarded target tissue to form proliferative tissue which is capable of forming somatic embryos;
(e) during the step of inducing, culturing the bombarded target tissue on selection media so as to select for embryogenic tissue which is transformed by the gene of interest;
(f) inducing transformed somatic embryos to develop from the selected embryogenic tissue; and (g) germinating and converting the transformed somatic embryos thus produced into clonal transgenic conifer plants.
(b) bombarding the target tissue by physically accelerating at the target tissue carrier particles which are much smaller than the cells of the target tissue, the carrier particles carrying copies of a genetic construction including at least one gene of interest;
(c) culturing the bombarded target tissue on preparation medium containing a sufficient amount of inorganic and organic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 150.0 mg/l of abscisic acid, about 10.0 to about 120.0 g/l of sugar, and up to about 0.5M of organic alcohol, for a sufficient period of time to allow the bombarded target tissue to recover from carrier particle insertion;
(d) inducing the bombarded target tissue to form proliferative tissue which is capable of forming somatic embryos;
(e) during the step of inducing, culturing the bombarded target tissue on selection media so as to select for embryogenic tissue which is transformed by the gene of interest;
(f) inducing transformed somatic embryos to develop from the selected embryogenic tissue; and (g) germinating and converting the transformed somatic embryos thus produced into clonal transgenic conifer plants.
12. The method of claim 1 which further comprises:
(a) culturing conifer target tissue selected from the group consisting of embryogenic tissue containing pre-stage 3 somatic embryos, pre-stage 3 somatic embryos, pre-stage 3 zygotic embryos, and combinations thereof, on preparation media containing a sufficient amount of inorganic and organic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 150.0 mg/l of abscisic acid, about 10.0 to about 120.0 g/l of sugar, and up to about 0.5M of organic alcohol, for a sufficient period of time to prepare the target tissue for bombardment by carrier particles;
(b) placing the prepared target tissue on a target surface;
(c) bombarding the prepared target tissue by physically accelerating at the prepared target tissue carrier particles which are much smaller than the cells of the target tissue, the carrier particles carrying copies of a genetic construction including at least one gene of interest;
(d) culturing the bombarded target tissue on preparation medium containing a sufficient amount of inorganic and organic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 150.0 mg/l of abscisic acid, about 10.0 to about 120.0 g/l of sugar, and up to about 0.5M of organic alcohol, for a sufficient period of time to allow the bombarded target tissue to recover from carrier particle insertion;
(e) inducing the bombarded target tissue to form proliferative tissue which is capable of forming somatic embryos;
(f) during the step of inducing, culturing the bombarded target tissue on selection media so as to select for embryogenic tissue which is transformed by the gene of interest;
(g) inducing transformed somatic embryos to develop from the selected embryogenic tissue; and (h) germinating and converting the transformed somatic embryos thus produced into clonal transgenic conifer plants.
(a) culturing conifer target tissue selected from the group consisting of embryogenic tissue containing pre-stage 3 somatic embryos, pre-stage 3 somatic embryos, pre-stage 3 zygotic embryos, and combinations thereof, on preparation media containing a sufficient amount of inorganic and organic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 150.0 mg/l of abscisic acid, about 10.0 to about 120.0 g/l of sugar, and up to about 0.5M of organic alcohol, for a sufficient period of time to prepare the target tissue for bombardment by carrier particles;
(b) placing the prepared target tissue on a target surface;
(c) bombarding the prepared target tissue by physically accelerating at the prepared target tissue carrier particles which are much smaller than the cells of the target tissue, the carrier particles carrying copies of a genetic construction including at least one gene of interest;
(d) culturing the bombarded target tissue on preparation medium containing a sufficient amount of inorganic and organic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 150.0 mg/l of abscisic acid, about 10.0 to about 120.0 g/l of sugar, and up to about 0.5M of organic alcohol, for a sufficient period of time to allow the bombarded target tissue to recover from carrier particle insertion;
(e) inducing the bombarded target tissue to form proliferative tissue which is capable of forming somatic embryos;
(f) during the step of inducing, culturing the bombarded target tissue on selection media so as to select for embryogenic tissue which is transformed by the gene of interest;
(g) inducing transformed somatic embryos to develop from the selected embryogenic tissue; and (h) germinating and converting the transformed somatic embryos thus produced into clonal transgenic conifer plants.
13. The method of claims 10, 11, and 12 wherein the sugar is a member selected from the group consisting of glucose, maltose, sucrose, and combinations thereof.
14. The method of claims 10, 11, and 12 wherein the organic alcohol is a member selected from the group consisting of glycerol, mannitol, sorbitol, polyethylene glycol, and combinations thereof.
15. The method of claims 10, 11, and 12 wherein the preparation medium further contains a gelling agent selected from the group consisting of about 6.0 to about 9.0 g/l of agar, about 1.75 to about 5.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 5.0 g/l of AGARGEL, and combinations thereof.
16. The method of claim 1 wherein the target tissue has been retrieved from cryopreservation.
17. The method of claim 1 wherein the bombarded target tissue is cryopreserved.
18. The method of claim 1 wherein the selected embryogenic tissue is cryopreserved.
19. The transformed embryogenic tissue of claim 1.
20. The transformed somatic embryos of claim 1.
21. The transgenic conifer plants of claim 1.
22. A method for genetically engineering conifers selected from the group consisting of the genus Pinus and Pinus interspecies hybrids, which comprises:
(a) placing a suitable conifer explant on culture initiation medium containing a sufficient amount of inorganic and organic nutrients, about 0.1 to about 5.0 mg/l of auxin, about 0.1 to about 1.0 mg/l of cytokinin, up to about 100.0 mg/l of abscisic acid, about 5.0 to about 100.0 g/l of sugar, and a gelling agent selected from the group consisting of about 2.5 to about 9.0 g/l of agar, about 0.5 to about 4.0 g/l of gellan gum, about 3.0 to about 10.0 g/l of agarose, about 1.5 to about 5.0 g/l of AGARGEL, and combinations thereof, for a sufficient amount of time under suitable environmental conditions to grow an embryogenic tissue culture containing pre-stage 3 somatic embryos;
(b) placing target tissue from the embryogenic tissue culture on a target surface, wherein the placed target tissue is selected from the group consisting of embryogenic tissue containing pre-stage 3 somatic embryos, pre-stage 3 somatic embryos, and combinations thereof;
(c) bombarding the target tissue by physically accelerating at the target tissue carrier particles which are much smaller than the cells of the target tissue, the carrier particles carrying copies of a genetic construction including at least one gene of interest;
(d) transferring the bombarded target tissue to selection medium so as to select for embryogenic tissue which is transformed by the gene of interest;
(e) transferring the transformed embryogenic tissue to embryo development medium containing a sufficient amount of inorganic and organic nutrients, about 5.0 mg/l to about 300.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, about 20.0 to about 70.0 g/l of sugar, and a gelling agent selected from the group consisting of about 6.0 to about 12.0 g/l of agar, about 1.75 to about 4.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 6.0 g/l of AGARGEL, and combinations thereof, for a sufficient time under suitable environmental conditions to develop transgenic stage 3 somatic embryos;
(f) separating the transgenic stage 3 somatic embryos from the development medium and partially drying the embryos by exposing the embryos to an atmosphere having a high relative humidity for a period of about 2 to about 5 weeks;
(g) transferring the partially dried transgenic embryos to germination medium containing a sufficient amount of organic and inorganic nutrients, up to about 10.0 g/l of activated carbon, about 20.0 to about 40.0 g/l of sugar, and a gelling agent selected from the group consisting of 6.0 to 9.0 g/l of agar, 1.75 to 3.50 g/l of gellan gum, 6.0 to 8.0 g/l of agarose, 3.5 to 5.0 g/l of AGARGEL, and combinations thereof, for a sufficient time under suitable environmental conditions to germinate the partially dried transgenic embryos;
(h) converting the germinated transgenic embryos into acclimatized transgenic conifer plants; and (i) field planting the acclimatized transgenic conifer plants.
(a) placing a suitable conifer explant on culture initiation medium containing a sufficient amount of inorganic and organic nutrients, about 0.1 to about 5.0 mg/l of auxin, about 0.1 to about 1.0 mg/l of cytokinin, up to about 100.0 mg/l of abscisic acid, about 5.0 to about 100.0 g/l of sugar, and a gelling agent selected from the group consisting of about 2.5 to about 9.0 g/l of agar, about 0.5 to about 4.0 g/l of gellan gum, about 3.0 to about 10.0 g/l of agarose, about 1.5 to about 5.0 g/l of AGARGEL, and combinations thereof, for a sufficient amount of time under suitable environmental conditions to grow an embryogenic tissue culture containing pre-stage 3 somatic embryos;
(b) placing target tissue from the embryogenic tissue culture on a target surface, wherein the placed target tissue is selected from the group consisting of embryogenic tissue containing pre-stage 3 somatic embryos, pre-stage 3 somatic embryos, and combinations thereof;
(c) bombarding the target tissue by physically accelerating at the target tissue carrier particles which are much smaller than the cells of the target tissue, the carrier particles carrying copies of a genetic construction including at least one gene of interest;
(d) transferring the bombarded target tissue to selection medium so as to select for embryogenic tissue which is transformed by the gene of interest;
(e) transferring the transformed embryogenic tissue to embryo development medium containing a sufficient amount of inorganic and organic nutrients, about 5.0 mg/l to about 300.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, about 20.0 to about 70.0 g/l of sugar, and a gelling agent selected from the group consisting of about 6.0 to about 12.0 g/l of agar, about 1.75 to about 4.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 6.0 g/l of AGARGEL, and combinations thereof, for a sufficient time under suitable environmental conditions to develop transgenic stage 3 somatic embryos;
(f) separating the transgenic stage 3 somatic embryos from the development medium and partially drying the embryos by exposing the embryos to an atmosphere having a high relative humidity for a period of about 2 to about 5 weeks;
(g) transferring the partially dried transgenic embryos to germination medium containing a sufficient amount of organic and inorganic nutrients, up to about 10.0 g/l of activated carbon, about 20.0 to about 40.0 g/l of sugar, and a gelling agent selected from the group consisting of 6.0 to 9.0 g/l of agar, 1.75 to 3.50 g/l of gellan gum, 6.0 to 8.0 g/l of agarose, 3.5 to 5.0 g/l of AGARGEL, and combinations thereof, for a sufficient time under suitable environmental conditions to germinate the partially dried transgenic embryos;
(h) converting the germinated transgenic embryos into acclimatized transgenic conifer plants; and (i) field planting the acclimatized transgenic conifer plants.
23. The method of claim 22 wherein the conifer is selected from the group consisting of Pinus taeda, Pinus serotina, Pinus palustris, Pinus elliottii, Pinus rigida, Pinus radiata, and hybrids thereof.
24. The method of claim 22 wherein the conifer explant is a member selected from the group consisting of immature zygotic embryos and megagametophytes containing immature zygotic embryos.
25. The method of claim 22 wherein the sugar is a member selected from the group consisting of glucose, maltose, sucrose, and combinations thereof.
26. The method of claim 22 wherein the carrier particles are microparticles between 0.2 and 2.0 microns in diameter.
27. The method of claim 22 wherein the selection medium contains a sufficient amount of organic and inorganic nutrients, a selection agent at a concentration which is toxic to non-transformed cells but for which the gene of interest confers resistance to transformed cells, up to 5.0 mg/l of auxin, up to 1.0 mg/l of cytokinin, up to about 30.0 mg/l of abscisic acid, and up to about 60.0 g/l of sugar.
28. The method of claim 22 wherein the selection medium contains a sufficient amount of organic and inorganic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 30.0 mg/l of abscisic acid, up to about 60.0 g/l of sugar, and wherein the selection medium lacks a component necessary for the growth of non-transformed cells but for which the gene of interest confers to transformed cells the ability to produce the lacking component.
29. The method of claim 22 wherein the selection medium contains a sufficient amount of organic and inorganic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 30.0 mg/l of abscisic acid, up to about 60.0 g/l of sugar, and wherein the selection medium contains a component necessary for the growth of cells in a form which cannot be utilized by non-transformed cells but for which the gene of interest confers to transformed cells the ability to utilize the necessary component.
30. The method of claim 22 wherein the selection medium contains a sufficient amount of organic and inorganic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 30.0 mg/l of abscisic acid, up to about 60.0 g/l of sugar, and wherein the selection medium allows preferential growth of transformed cells containing the gene of interest.
31. The method of claims 27, 28, 29, and 30 wherein the selection medium further contains a gelling agent selected from the group consisting of about 6.0 to about 9.0 g/l of agar, about 1.75 to about 4.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 5.0 g/l of AGARGEL, and combinations thereof.
32. The method of claim 22 which further comprises:
(1) the addition of up to about 100.0 g/l of polyethylene glycol to the embryo development medium(e); and (2) transfernng the transgenic stage 3 embryos from the embryo development medium to a second development medium containing a sufficient amount of inorganic and organic nutrients, about 5.0 mg/l to about 300.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, up to about 100.0 g/l of polyethylene glycol, and about 20.0 to about 70.0 g/l of sugar, for a sufficient time under suitable environmental conditions to further develop the transgenic stage 3 somatic embryos prior to partially drying the embryos.
(1) the addition of up to about 100.0 g/l of polyethylene glycol to the embryo development medium(e); and (2) transfernng the transgenic stage 3 embryos from the embryo development medium to a second development medium containing a sufficient amount of inorganic and organic nutrients, about 5.0 mg/l to about 300.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, up to about 100.0 g/l of polyethylene glycol, and about 20.0 to about 70.0 g/l of sugar, for a sufficient time under suitable environmental conditions to further develop the transgenic stage 3 somatic embryos prior to partially drying the embryos.
33. The method of claim 22 which further comprises:
(1) the addition of up to about 100.0 g/l of polyethylene glycol to the embryo development medium (e); and (2) transferring the transgenic stage 3 embryos from the embryo development medium to a second development medium containing a sufficient amount of inorganic and organic nutrients, up to about 100.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, up to about 100.0 g/l of polyethylene glycol, and about 20.0 to about 70.0 g/l of sugar, for a period of about 2 to about 12 weeks at a temperature in the range of about 0°C to about 10°C under suitable environmental conditions to maintain the viability of the transgenic stage 3 somatic embryos prior to partially drying the embryos.
(1) the addition of up to about 100.0 g/l of polyethylene glycol to the embryo development medium (e); and (2) transferring the transgenic stage 3 embryos from the embryo development medium to a second development medium containing a sufficient amount of inorganic and organic nutrients, up to about 100.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, up to about 100.0 g/l of polyethylene glycol, and about 20.0 to about 70.0 g/l of sugar, for a period of about 2 to about 12 weeks at a temperature in the range of about 0°C to about 10°C under suitable environmental conditions to maintain the viability of the transgenic stage 3 somatic embryos prior to partially drying the embryos.
34. The method of claim 22 wherein the target tissue from the embryogenic tissue culture, prior to the bombardment by the carrier particles, is cultured on preparation media containing a sufficient amount of inorganic and organic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 150.0 mg/l of abscisic acid, about 10.0 to about 120.0 g/l of sugar, and up to about 0.5M of organic alcohol, for a sufficient period of time to prepare the target tissue for bombardment by carrier particles.
35. The method of claim 22 wherein the bombarded target tissue, prior to being transferred to the selection medium, is cultured on preparation media containing a sufficient amount of inorganic and organic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 150.0 mg/l of abscisic acid, about 10.0 to about 120.0 g/l of sugar, and up to about 0.5M of organic alcohol, for a sufficient period of time to allow the bombarded target tissue to recover from carrier particle insertion.
36. The method of claims 34 and 35 wherein the organic alcohol is a member selected from the group consisting of glycerol, mannitol, sorbitol, polyethylene glycol, and combinations thereof.
37. The method of claims 34 and 35 wherein the preparation medium further contains a gelling agent selected from the group consisting of about 6.0 to about 9.0 g/l of agar, about 1.75 to about 5.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 5.0 g/l of AGARGEL, and combinations thereof.
38. The method of claim 22 wherein the embryogenic tissue culture from the culture initiation medium is cultured on culture maintenance medium containing a sufficient amount of inorganic and organic nutrients, about 0.1 to about 5.0 mg/l of auxin, about 0.1 to about 1.0 mg/l of cytokinin, up to about 100.0 mg/l of abscisic acid, up to about 10.0 g/l of activated carbon, and about 10.0 to about 40.0 g/l of sugar for a sufficient time under suitable environmental conditions to grow the embryogenic tissue culture containing pre-stage 3 somatic embryos.
39. The method of claim 22 wherein the bombarded target tissue is cultured on culture maintenance media containing a sufficient amount of inorganic and organic nutrients, about 0.1 to about 5.0 mg/l of auxin, about 0.1 to about 1.0 mg/l of cytokinin, up to about 100.0 mg/l of abscisic acid, up to about 10.0 g/l of activated carbon, and about 10.0 to about 40.0 g/l of sugar for a sufficient time under suitable environmental conditions to grow the bombarded target tissue.
40. The method of claim 22 wherein the transformed embryogenic tissue is cultured on culture maintenance media containing a sufficient amount of inorganic and organic nutrients, about 0.1 to about 5.0 mg/l of auxin, about 0.1 to about 1.0 mg/l of cytokinin, up to about 100.0 mg/l of abscisic acid, up to about 10.0 g/l of activated carbon, and about 10.0 to about 40.0 g/l of sugar for a sufficient time under suitable environmental conditions to grow the transformed embryogenic tissue.
41. The method of claims 38, 39, and 40 and further contains a gelling agent selected from the group consisting of about 6.0 to about 9.0 g/l of agar, about 1.75 to about 4.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 5.0 g/l of AGARGEL, and combinations thereof.
42. The method of claim 22 wherein the embryogenic tissue culture from the culture initiation medium is cultured on embryo development medium containing a sufficient amount of inorganic and organic nutrients, about 5.0 mg/l to about 300.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, about 20.0 to about 70.0 g/l of sugar, and a gelling agent selected from the group consisting of about 6.0 to about 12.0 g/l of agar, about 1.75 to about 4.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 6.0 g/l of AGARGEL, and combinations thereof, for a sufficient time under suitable environmental conditions to prepare the target tissue for carrier particle bombardment.
43. The method of claim 22 wherein the target tissue has been retrieved from cryopreservation.
44. The method of claim 22 wherein the bombarded target tissue is cryopreserved.
45. The method of claim 22 wherein the transformed embryogenic tissue is cryopreserved.
46. The transformed embryogenic tissue of claim 22.
47. The transgenic somatic embryos of claim 22.
48. The transgenic conifer plants of claim 22.
49. A method for genetically engineering conifers selected from the group consisting of the genus Pinus and Pinus interspecies hybrids, which comprises:
(a) placing conifer target tissue selected from the group consisting of pre-stage 3 zygotic embryos, tissues extruded from immature megagameophytes which contain pre-stage 3 zygotic embryos, and combinations thereof, on a target surface;
(b) bombarding the target tissue by physically accelerating at the target tissue carrier particles which are much smaller than the cells of the target tissue, the carrier particles carrying copies of a genetic construction including at least one gene of interest;
(c) transferring the bombarded target tissue to selection medium so as to select for embryogenic tissue which is transformed by the gene of interest;
(d) transferring the transformed embryogenic tissue to embryo development medium containing a sufficient amount of inorganic and organic nutrients, about 5.0 mg/l to about 300.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, about 20.0 to about 70.0 g/l of sugar, and a gelling agent selected from the group consisting of about 6.0 to about 12.0 g/l of agar, about 1.75 to about 4.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 6.0 g/l of AGARGEL, and combinations thereof, for a sufficient time under suitable environmental conditions to develop transgenic stage 3 somatic embryos;
(e) separating the transgenic stage 3 somatic embryos from the development medium and partially drying the embryos by exposing the embryos to an atmosphere having a high relative humidity for a period of about 2 to about 5 weeks;
(f) transferring the partially dried transgenic embryos to germination medium containing a sufficient amount of organic and inorganic nutrients, up to about 10.0 g/l of activated carbon, about 20.0 to about 40.0 g/l of sugar, and a gelling agent selected from the group consisting of 6.0 to 9.0 g/l of agar, 1.75 to 3.50 g/l of gellan gum, 6.0 to 8.0 g/l of agarose, 3.5 to 5.0 g/l of AGARGEL, and combinations thereof, for a sufficient time under suitable environmental conditions to germinate the partially dried transgenic embryos;
(g) converting the germinated transgenic embryos into acclimatized transgenic conifer plants; and (h) field planting the acclimatized transgenic conifer plants.
(a) placing conifer target tissue selected from the group consisting of pre-stage 3 zygotic embryos, tissues extruded from immature megagameophytes which contain pre-stage 3 zygotic embryos, and combinations thereof, on a target surface;
(b) bombarding the target tissue by physically accelerating at the target tissue carrier particles which are much smaller than the cells of the target tissue, the carrier particles carrying copies of a genetic construction including at least one gene of interest;
(c) transferring the bombarded target tissue to selection medium so as to select for embryogenic tissue which is transformed by the gene of interest;
(d) transferring the transformed embryogenic tissue to embryo development medium containing a sufficient amount of inorganic and organic nutrients, about 5.0 mg/l to about 300.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, about 20.0 to about 70.0 g/l of sugar, and a gelling agent selected from the group consisting of about 6.0 to about 12.0 g/l of agar, about 1.75 to about 4.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 6.0 g/l of AGARGEL, and combinations thereof, for a sufficient time under suitable environmental conditions to develop transgenic stage 3 somatic embryos;
(e) separating the transgenic stage 3 somatic embryos from the development medium and partially drying the embryos by exposing the embryos to an atmosphere having a high relative humidity for a period of about 2 to about 5 weeks;
(f) transferring the partially dried transgenic embryos to germination medium containing a sufficient amount of organic and inorganic nutrients, up to about 10.0 g/l of activated carbon, about 20.0 to about 40.0 g/l of sugar, and a gelling agent selected from the group consisting of 6.0 to 9.0 g/l of agar, 1.75 to 3.50 g/l of gellan gum, 6.0 to 8.0 g/l of agarose, 3.5 to 5.0 g/l of AGARGEL, and combinations thereof, for a sufficient time under suitable environmental conditions to germinate the partially dried transgenic embryos;
(g) converting the germinated transgenic embryos into acclimatized transgenic conifer plants; and (h) field planting the acclimatized transgenic conifer plants.
50. The method of claim 49 wherein the conifer is selected from the group consisting of Pinus taeda, Pinus serotina, Pinus palustris, Pinus elliottii, Pinus rigida, Pinus radiata, and hybrids thereof.
51. The method of claim 49 wherein the conifer target tissue consists of immature megagameophytes which contain pre-stage 3 zygotic embryos; the bombarded target tissue is cultured to encourage the extrusion of the bombarded pre-stage 3 zygotic embryos; and the extruded bombarded pre-stage 3 zygotic embryos are transferred to selection medium for selection of transformed cells.
52. The method of claim 49 wherein the sugar is a member selected from the group consisting of glucose, maltose, sucrose, and combinations thereof.
53. The method of claim 49 wherein the carrier particles are microparticles between 0.2 and 2.0 microns in diameter.
54. The method of claim 49 wherein the selection medium contains a sufficient amount of organic and inorganic nutrients, a selection agent at a concentration which is toxic to non-transformed cells but for which the gene of interest confers resistance to transformed cells, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 30.0 mg/l of abscisic acid, and up to about 60.0 g/l of sugar.
55. The method of claim 49 wherein the selection medium contains a sufficient amount of organic and inorganic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 30.0 mg/l of abscisic acid, up to about 60.0 g/l of sugar, and wherein the selection medium lacks a component necessary for the growth of non-transformed cells but for which the gene of interest confers to transformed cells the ability to produce the lacking component.
56. The method of claim 49 wherein the selection medium contains a sufficient amount of organic and inorganic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 30.0 mg/l of abscisic acid, up to about 60.0 g/l of sugar, and wherein the selection medium contains a component necessary for the growth of cells in a form which cannot be utilized by non-transformed cells but for which the gene of interest confers to transformed cells the ability to utilize the necessary component.
57. The method of claim 49 wherein the selection medium contains a sufficient amount of organic and inorganic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 30.0 mg/l of abscisic acid, up to about 60.0 g/l of sugar, and wherein the selection medium allows preferential growth of transformed cells containing the gene of interest.
58. The method of claims 54, 55, 56, and 57 wherein the selection medium further contains a gelling agent selected from the group consisting of about 6.0 to about 9.0 g/l of agar, about 1.75 to about 4.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 5.0 g/l of AGARGEL, and combinations thereof.
59. The method of claim 49 which further comprises:
(1) the addition of up to about 100.0 g/l of polyethylene glycol to the embryo development medium (e); and (2) transferring the transgenic stage 3 embryos from the embryo development medium to a second development medium containing a sufficient amount of inorganic and organic nutrients, about 5.0 mg/l to about 300.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, up to about 100.0 g/l of polyethylene glycol, and about 20.0 to about 70.0 g/l of sugar, for a sufficient time under suitable environmental conditions to further develop the transgenic stage 3 somatic embryos prior to partially drying the embryos.
(1) the addition of up to about 100.0 g/l of polyethylene glycol to the embryo development medium (e); and (2) transferring the transgenic stage 3 embryos from the embryo development medium to a second development medium containing a sufficient amount of inorganic and organic nutrients, about 5.0 mg/l to about 300.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, up to about 100.0 g/l of polyethylene glycol, and about 20.0 to about 70.0 g/l of sugar, for a sufficient time under suitable environmental conditions to further develop the transgenic stage 3 somatic embryos prior to partially drying the embryos.
60. The method of claim 49 which further comprises:
(1) the addition of up to about 100.0 g/l of polyethylene glycol to the embryo development medium (e); and (2) transferring the transgenic stage 3 embryos from the embryo development medium to a second development medium containing a sufficient amount of inorganic and organic nutrients, up to about 100.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, up to about 100.0 g/l of polyethylene glycol, and about 20.0 to about 70.0 g/l of sugar, for a period of about 2 to about 12 weeks at a temperature in the range of about 0°C to about 10°C under suitable environmental conditions to maintain the viability of the transgenic stage 3 somatic embryos prior to partially drying the embryos.
(1) the addition of up to about 100.0 g/l of polyethylene glycol to the embryo development medium (e); and (2) transferring the transgenic stage 3 embryos from the embryo development medium to a second development medium containing a sufficient amount of inorganic and organic nutrients, up to about 100.0 mg/l of abscisic acid with the continued maintenance of the abscisic acid concentration, up to about 10.0 g/l of activated carbon, up to about 100.0 g/l of polyethylene glycol, and about 20.0 to about 70.0 g/l of sugar, for a period of about 2 to about 12 weeks at a temperature in the range of about 0°C to about 10°C under suitable environmental conditions to maintain the viability of the transgenic stage 3 somatic embryos prior to partially drying the embryos.
61. The method of claim 49 wherein the target tissue from the embryogenic tissue culture, prior to the bombardment by the carrier particles, is cultured on preparation media containing a sufficient amount of inorganic and organic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 150.0 mg/l of abscisic acid, about 10.0 to about 120.0 g/l of sugar, and up to about 0.5M of organic alcohol, for a sufficient period of time to prepare the target tissue for bombardment by carrier particles.
62. The method of claim 49 wherein the bombarded target tissue, prior to being transferred to the selection medium, is cultured on preparation media containing a sufficient amount of inorganic and organic nutrients, up to about 5.0 mg/l of auxin, up to about 1.0 mg/l of cytokinin, up to about 150.0 mg/l of abscisic acid, about 10.0 to about 120.0 g/l of sugar, and up to about 0.5M of organic alcohol, for a sufficient period of time to allow the bombarded target tissue to recover from carrier particle insertion.
63. The method of claims 61 and 62 wherein the organic alcohol is a member selected from the group consisting of glycerol, mannitol, sorbitol, polyethylene glycol, and combinations thereof.
64. The method of claims 61 and 62 wherein the preparation medium further contains a gelling agent selected from the group consisting of about 6.0 to about 9.0 g/l of agar, about 1.75 to about 5.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 5.0 g/l of AGARGEL, and combinations thereof.
65. The method of claim 49 wherein the bombarded target tissue is cultured on culture maintenance media containing a sufficient amount of inorganic and organic nutrients, about 0.1 to about 5.0 mg/l of auxin, about 0.1 to about 1.0 mg/l of cytokinin, up to about 100.0 mg/l of abscisic acid, up to about 10.0 g/l of activated carbon, and about 10.0 to about 40.0 g/l of sugar for a sufficient time under suitable environmental conditions to grow the bombarded target tissue.
66. The method of claim 49 wherein the transformed embryogenic tissue is cultured on culture maintenance media containing a sufficient amount of inorganic and organic nutrients, about 0.1 to about 5.0 mg/l of auxin, about 0.1 to about 1.0 mg/l of cytokinin, up to about 100.0 mg/l of abscisic acid, up to about 10.0 g/l of activated carbon, and about 10.0 to about 40.0 g/l of sugar for a sufficient time under suitable environmental conditions to grow the transformed embryogenic tissue.
67. The method of claims 65 and 66 wherein the culture maintenance media further contains a gelling agent selected from the group consisting of about 6.0 to about 9.0 g/l of agar, about 1.75 to about 4.0 g/l of gellan gum, about 6.0 to about 8.0 g/l of agarose, about 3.5 to about 5.0 g/l of AGARGEL, and combinations thereof.
68. The method of claim 49 wherein the target tissue has been retrieved from cryopreservation.
69. The method of claim 49 wherein the bombarded target tissue is cryopreserved.
70. The method of claim 49 wherein the transformed embryogenic tissue is cryopreserved.
71. The transformed embryogenic tissue of claim 49.
72. The transgenic somatic embryos of claim 49.
73. The transgenic conifer plants of claim 49.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US8796698P | 1998-06-04 | 1998-06-04 | |
| US60/087,966 | 1998-06-04 | ||
| US09/318,136 US6518485B1 (en) | 1998-06-04 | 1999-05-25 | Particle-mediated conifer transformation |
| US09/318,136 | 1999-05-25 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2274037A1 true CA2274037A1 (en) | 1999-12-04 |
| CA2274037C CA2274037C (en) | 2010-12-14 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2274037A Expired - Fee Related CA2274037C (en) | 1998-06-04 | 1999-06-03 | Particle-mediated conifer transformation |
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| US (1) | US6518485B1 (en) |
| AR (1) | AR020593A1 (en) |
| AU (1) | AU779492B2 (en) |
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| CA (1) | CA2274037C (en) |
| NZ (1) | NZ336149A (en) |
| ZA (1) | ZA993748B (en) |
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| US6518485B1 (en) | 1998-06-04 | 2003-02-11 | Westvaco Corporation | Particle-mediated conifer transformation |
| US7365186B2 (en) * | 2002-11-22 | 2008-04-29 | Arborgen, Llc | Vascular-preferred promoter sequences and uses thereof |
| WO2005001101A1 (en) * | 2003-06-03 | 2005-01-06 | University Of Georgia Research Foundation, Inc. | Conditional sterility in plants |
| US7402428B2 (en) | 2004-09-22 | 2008-07-22 | Arborgen, Llc | Modification of plant lignin content |
| US7456338B2 (en) | 2004-09-22 | 2008-11-25 | Arborgen Llc | Modification of plant lignin content |
| US7799906B1 (en) | 2004-09-22 | 2010-09-21 | Arborgen, Llc | Compositions and methods for modulating lignin of a plant |
| US20060101536A1 (en) * | 2004-11-05 | 2006-05-11 | Arborgen, Llc | Eucalyptus urophylla transformation and regeneration |
| BR112016003561B8 (en) * | 2013-08-22 | 2022-11-01 | Du Pont | METHOD FOR PRODUCING A GENETIC MODIFICATION, METHOD FOR INTRODUCING A POLYNUCLEOTIDE OF INTEREST INTO THE GENOME OF A PLANT, METHOD FOR EDITING A SECOND GENE INTO A PLANT GENOME AND METHOD FOR GENERATING A GLYPHOSATE-RESISTANT CORN PLANT |
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| NL8300699A (en) | 1983-02-24 | 1984-09-17 | Univ Leiden | METHOD FOR BUILDING FOREIGN DNA INTO THE NAME OF DIABIC LOBAL PLANTS; METHOD FOR PRODUCING AGROBACTERIUM TUMEFACIENS BACTERIEN; STABLE COINTEGRATE PLASMIDS; PLANTS AND PLANT CELLS WITH CHANGED GENETIC PROPERTIES; PROCESS FOR PREPARING CHEMICAL AND / OR PHARMACEUTICAL PRODUCTS. |
| US4672035A (en) * | 1984-03-16 | 1987-06-09 | Research Corporation | Controlled regeneration of cotton plants from tissue culture |
| US4945050A (en) | 1984-11-13 | 1990-07-31 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues and apparatus therefor |
| US4886937A (en) | 1985-05-20 | 1989-12-12 | North Carolina State University | Method for transforming pine |
| US5122466A (en) | 1989-06-13 | 1992-06-16 | North Carolina State University | Ballistic transformation of conifers |
| CA2075135A1 (en) | 1991-08-02 | 1993-02-03 | David E. Ellis | Particle-mediated transformation of gymnosperms |
| US5565355A (en) | 1991-12-19 | 1996-10-15 | New Zealand Forest Research Institute Limited | Growth medium |
| WO1994007902A1 (en) | 1992-10-05 | 1994-04-14 | North Carolina State University | Method for increasing expression and reducing expression variability of foreign genes in plant cells |
| US5506136A (en) | 1993-10-21 | 1996-04-09 | Westvaco Corporation | Method for regeneration of coniferous plants by somatic embryogenesis |
| US5413930A (en) | 1993-10-21 | 1995-05-09 | Westvaco Corporation | Method for regeneration of coniferous plants by somatic embryogenesis |
| US5491090A (en) | 1994-02-09 | 1996-02-13 | Westvaco Corporation | Embryogenic coniferous liquid suspension cultures |
| AU6141796A (en) | 1995-06-26 | 1997-01-30 | New Zealand Forest Research Institute Limited | Stable transformation of undifferentiated conifer cells |
| US5677185A (en) | 1996-05-14 | 1997-10-14 | Westvaco Corporation | Method for regeneration of coniferous plants by somatic embryogenesis in culture media containing abscisic acid |
| US5731203A (en) | 1996-06-14 | 1998-03-24 | Westvaco Corporation | Method for regeneration of coniferous plants by somatic embryogenesis |
| US5731191A (en) | 1996-12-20 | 1998-03-24 | Westvaco Corporation | Method for regeneration of coniferous plants by somatic embryogenesis employing polyethylene glycol |
| US5731204A (en) | 1996-12-20 | 1998-03-24 | Westvaco Corporation | Method for regeneration of coniferous plants by somatic embryogenesis employing polyethylene glycol |
| US6518485B1 (en) | 1998-06-04 | 2003-02-11 | Westvaco Corporation | Particle-mediated conifer transformation |
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- 1999-06-03 CA CA2274037A patent/CA2274037C/en not_active Expired - Fee Related
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- 1999-06-04 NZ NZ336149A patent/NZ336149A/en not_active IP Right Cessation
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| AU3316599A (en) | 1999-12-16 |
| BR9901778A (en) | 2000-05-09 |
| CA2274037C (en) | 2010-12-14 |
| NZ336149A (en) | 2000-10-27 |
| ZA993748B (en) | 1999-12-03 |
| AR020593A1 (en) | 2002-05-22 |
| US6518485B1 (en) | 2003-02-11 |
| AU779492B2 (en) | 2005-01-27 |
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