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United States Patent 19 11 Patent Number: 5,821,126 Durzan Et Al

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United States Patent 19 11 Patent Number: 5,821,126 Durzan Et Al USOO5821126A United States Patent 19 11 Patent Number: 5,821,126 Durzan et al. (45) Date of Patent: Oct. 13, 1998 54 METHOD FOR CLONAL PROPAGATION OF 52 U.S. Cl. .......................... 435/422; 435/420; 435/430; GYMNOSPERMS BY SOMATIC 435/430.1; 435/431; 47/57.6; 47/58 POLYEMBRYOGENESIS 58 Field of Search ......................... 435/240.45, 240.48, 435/240.49, 240.54, 240.46, 420, 422, 430, 75 Inventors: Don J. Durzan, Davis, Calif.; Pramod 430.1, 431; 800/DIG. 49–51; 47/57.6, 58 K. Gupta, Federal Way, Wash. 56) References Cited 73 Assignee: The Regents of the University of California, Oakland, Calif. PUBLICATIONS Hakman et al. 1985. Plant Science 38:53–59. 21 Appl. No.: 398,060 Hakman et al. 1985. J. Plant Physiol. 121(2):149-158. Krogstrup, P. 1986. Can. J. For. Res. 16:664-668. 22 Filed: Mar. 3, 1995 Gupta et al. 1987. Bio/Technology 5(2): 147-151. Related U.S. Application Data Primary Examiner David T. Fox Attorney, Agent, or Firm-Hana Verny 63 Continuation-in-part of Ser. No. 908,958, Jul. 6, 1992, abandoned, and Ser. No. 876,695, Apr. 28, 1992, abandoned, 57 ABSTRACT whichabandoned, is a continuation which is a continuation of Ser. No. of701,597, Ser. No. May 537,863, 13, 1991, Jun. A method for clonal propagation by Somatic polyembryo 12, 1990, abandoned, which is a continuation of Ser. No. genesis. The method allows for clonal propagation of 65.610, Jun. 22, 1987, abandoned, which is a continuation embryonal Suspensor mass resulting in true-to-type Suspen SF o, SS, E. N. ship's Sor development of the conifer embryo leading to develop 13,e. 1989,TY.O. abandoned,958, is a continuationsaid Ser. No. oI 932,719. Ser. No. U95, Nov. ment of plantlets and plants. 51) Int. Cl. ................................ A01H 4/00; AO1C 1/06 28 Claims, 8 Drawing Sheets U.S. Patent Oct. 13, 1998 Sheet 1 of 8 5,821,126 IREE IMMATURE OR MAILURE SEED FIG. 1 EXPLANT SOURCES LEAVES APICAL MERISTEM EXCISED d2 IISSUE OR CULTURED 2 NUCELLAR NEW GENE ISSUE RAITOW CULTURING CULTURED 3. CELLS FROM A CELLS FROM ff B EN 4 ENEED CALLUS SOMATIC DISCARDED EMBRYOS 12 5 13 EMBRYO MATURATION DESSICATION EMBRYOGENIC CELL SUSPENSION OR SEMISOLID CRYOGENIC PLAIES ENCAP LA/TOW FREE NUCLEAR Roots 71 STAGE WITH 15 NUCLEAR MIGRATION BLEEDING, f5 9 IESI, " council 8 PLANINGS, PLAWINGS PROEMBRYO AND EARLY AWD EARLY SELECTION EBRYOS EMBRYO WITH IW ESM REPEATED DIVISION COLD EARLY CLEAVAGE EMBRYOS POLY - 10 IW ESM EMBRYONIC MASS U.S. Patent Oct. 13, 1998 Sheet 2 of 8 5,821,126 IG. 2A FIG. 2B s : 8 8 .8 U.S. Patent Oct. 13, 1998 Sheet 3 of 8 5,821,126 FIG. 2E FIG. 2G FIG. 2 U.S. Patent Oct. 13, 1998 Sheet 4 of 8 5,821,126 FIG. 3A FIG. 3C FIG. 3D U.S. Patent Oct. 13, 1998 Sheet 5 of 8 5,821,126 FIG. 3E FIG, 3G FIG. 3 U.S. Patent Oct. 13, 1998 Sheet 6 of 8 5,821,126 U.S. Patent Oct. 13, 1998 Sheet 7 of 8 5,821,126 FIG. 4F U.S. Patent Oct. 13, 1998 Sheet 8 of 8 5,821,126 FIG. 5 100 100 proembryo CS& 75 &Šs 75 S S. s 50 s 50 S. N &s 25 As 25 O O 0.5 5.0 20 0 O 0.5 5.0 20 2, 4-D (ppm) myo-INOSITOL (ppm) 100 f00 O proembryo Šs 75 Šs 75 S S S 50 SS. 50 S. s as 25 &e 25 O O O 0.5 5.0 20 0 0.5 50 20 CASEIN HYDROLYSATE (opm) ABSCISIC ACID (ppm) N-- 5,821,126 1 2 METHOD FOR CLONAL PROPAGATION OF copies of the embryo to be cloned and this would enable a GYMNOSPERMS BY SOMATIC wider testing of genotype performance over a wider range of POLYEMBRYOGENESIS environments. This cannot be done with one embryo from one Seed. This application is a continuation-in-part of U.S. Ser. No. 5 While previously simple Somatic polyembryogenesis 07/908,958 filed on Jul. 6, 1992, now abandoned, which is from callus of mature Sugar pine embryos was described a continuation of U.S. application Ser. No. 07/436,095, filed (Biotechnology, 4:643 (1986), a true-to-conifer-type somatic on Nov. 13, 1989, now abandoned, which is a continuation polyembryogenesis that proceeds from a proliferating of U.S. Ser. No. 06/932,719 filed on Nov. 19, 1986 now embryonal Suspensor cell mass was not reported before this abandoned. This application is also a continuation-in-part of invention was developed. U.S. application Ser. No. 07/876,695, filed on Apr. 28, 1992, Previous attempts to provide true-to-type Somatic poly now abandoned, which is a continuation of U.S. Ser. No. embryogenetic clonal propagation were not Successful. 07/701,597, filed on May 13, 1991, now abandoned, which These previous attempts for clonal propagation with Somatic is a continuation of U.S. Ser. No. 07/537,863 filed on Jun. cells did not produce and did not report embryonal Suspen 12, 1990, now abandoned, which is a continuation of 15 Sor mass development or true-to-type and Sequential devel 07/065,610 filed on Jun. 22, 1987, now abandoned, which is opment characteristics of the conifer embryo leading to a continuation-in-part of U.S. Ser. No. 06/932,719 filed on plantlets able to grow in Soil. Nov. 19, 1986, now abandoned. The earliest attempts to achieve Somatic embryogenesis in This invention was made with government Support under gymnosperms were described in Canadian Department of Grant NOS. PSW83-0038 CA and 84-0011 CA with the Forestry and Rural Development, Res. Rpts, 24:30 (1970)., United States Department of Agriculture and the University Abstr. Comm. Inst. For Fenn., 75:16 (1971); Proc. 50th Ann. of California. The government has certain rights in this Conf. Appleton, Wis., May 8–10, pp. 36-60 (1978), Proc. V. invention. Intl. Congr. Plant Cell and Tissue Culture, July 11-16, BACKGROUND OF THE INVENTION Tokyo, Japan, pp. 113-114 (1982); and U.S. Pat. No. 4,217, 25 730. Although these attempts were made since around 1970, 1. Field of the Invention none of these publications reported clonal Somatic or game The current invention concerns a method for clonal propa tophytic polyembryogenesis or true-to-conifer-type embry gation of gymnosperms by Somatic polyembryogenesis. In onic development. particular, the invention concerns a method for production of The earlier publications reported appearance of embryo embryonal Suspensor mass able to develop into a generation and Suspensor-like Structures in cell Suspensions of embry of gymnosperm clonal Somatic plant proembryos. The onic tissueS of white Spruce and jack pine in undefined method allows clonal propagation of Somatic cells resulting media, Res. Rpts. (1970) supra Plant Sci. Lett., 38:53–59 in true-to-type development of the conifer embryo leading to (1985) reported Somatic embryogenesis in a Norway spruce development of plantlets and plants. Additionally, the inven 35 “callus” using tissue from immature Seeds and Can. J. tion concerns cloning gymnosperm embryos, propagating Forestry Res., 15:1088–1091 (1985) reported the same in plantlets, trees, encapsulating embryos, Storing embryos Larch. None of these others, however, discovered the cel over a long period of time and diagnosing the developmental lular origin of the proceSS for production of or achieved Stages and condition of plant cells by Staining and microS true-to-type clonal Somatic or gametophytic polyembryos, copy. nor did they recognize or achieve the Specific attributes of 2. Background Art and Related Art Disclosures 40 cleavage polyembryony and the polyembryonic multiplica Somatic polyembryogenesis for clonal propagation of tion proceSS as described herein. Also, none of the reported gymnosperm, if achieved, would have far reaching effect on research generated Somatic embryos in any Significant num clonal forestry and agriculture development. ber. If successful, Somatic polyembryogenesis (SPE) would 45 Similarly, none of the publication disclosed diagnostic allow propagation of Seeds obtained from a Selected croSS of tests which could distinguish the developmental Stages of elite parents especially in advanced breeding programs plant embryogenesis. As a diagnostic tool, Biotech., 4:763 where the progeny are expected to be Superior to the average (1984) reported diagnostic color test for Somatic embryo of the population. The Selection and rescue of viable genesis in Prunus in cell Suspensions. Chromosome Tech embryonal Suspensor masses or of explants from elite tree 50 niques. Theory and Practice, Frakenham Press, Ltd., minimizes the introduction of undesirable traits introduced Norfolk, page 121, (1980), and J. Exp. Bot., 22:756-758 by introgression of foreign pollen or mutations that occur in (1971) described Staining methods for examining cells. Sexual reproduction. However, none of these articles utilized the use of chromatin Additionally, SPE would allow the controlled mainte or glycoprotein and viability test Stains for determining the nance and multiplications on demand of the genotype by cell 55 free nuclear and early developmental Stages of plant and tissue culture technology. This would remove the con embryogenesis. Straints to tree production where Seed production varies year One of the many difficulties in propagating Species of to year with Some years giving no Seed at all. The controlled gymnosperm plants has been a lack of preservation tech culture of cells and mass production of embryos would niques which would preserve the embryos in a viable State further enable convenient manipulation (genetic and non 60 even after a long term storage.
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