US20020188965A1 - Methods of transforming plants - Google Patents

Methods of transforming plants Download PDF

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US20020188965A1
US20020188965A1 US10/121,200 US12120002A US2002188965A1 US 20020188965 A1 US20020188965 A1 US 20020188965A1 US 12120002 A US12120002 A US 12120002A US 2002188965 A1 US2002188965 A1 US 2002188965A1
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cell
method
plant
haploid
homozygous
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Zou-Yu Zhao
Dennis Bidney
Evan Elsing
Michael Miller
Xinli Wu
William Gordon-Kamm
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Pioneer Hi Bred International Inc
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10Seeds
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/829Female sterility

Abstract

Methods for producing homozygous plants, seeds, and plant cells are provided. Also provided are methods of transformation.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application claims priority to U.S. Ser. No. 60/285,265 the disclosure of which is incorporated herein by reference.[0001]
  • FIELD OF THE INVENTION
  • The present invention relates to the field of genetic engineering of plants and to methods for introducing traits into plants. [0002]
  • BACKGROUND OF THE INVENTION
  • Many current transformation technologies produce mainly heterozygous transgenic plants. However, homozygous transgenic plants are basic for product development and commercialization of plants. To obtain homozygous transgenic plants requires several generations of self-pollination and segregation analysis. This is an inefficient use of labor and time resources. It would therefore be useful to develop a method to reduce hand pollination steps normally required to obtain a homozygous transgenic plant. [0003]
  • DETAILED DESCRIPTION OF THE INVENTION
  • As used herein “Growth Stimulation Polynucleotides” include polynucleotides whose encoded products stimulate growth either through triggering developmental programs (i.e. embryogenesis, meristem formation, meristem maintenance, etc) or through stimulating the cell cycle. [0004]
  • As used herein “Transformation” includes stable transformation and transient transformation unless indicated otherwise. [0005]
  • As used herein “Stable Transformation” refers to the transfer of a nucleic acid fragment into a genome of a host organism (this includes both nuclear and organelle genomes) resulting in genetically stable inheritance. In addition to traditional methods, stable transformation includes the alteration of gene expression by any means including chimerplasty or transposon insertion. [0006]
  • As used herein “Transient Transformation” refers to the transfer of a nucleic acid fragment or protein into the nucleus (or DNA-containing organelle) of a host organism resulting in gene expression without integration and stable inheritance. [0007]
  • As used herein, “nucleic acid” includes deoxyribonucleotide or ribonucleotide polymer, or chimeras thereof, in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). [0008]
  • As used herein, the term “plant” includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. “Plant cell”, as used herein includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The class of plants which can be used in the methods of the invention include both monocotyledonous and dicotyledonous plants. [0009]
  • Methods for obtaining homozygous plants, plant cells, and seeds are provided. Also provided are methods for obtaining haploid embryos and seeds and methods for increasing chromosomal doubling. The methods comprise contacting haploid cells with a chromosome doubling agent and providing a growth stimulation protein. The methods also comprise crossing a selected plant and an inducer line to produce haploid embryos or seeds while providing a growth stimulation polynucleotide. Other methods comprise crossing a selected plant and an inducer line to produce a haploid cell, providing a growth stimulation polynucleotide, and treating the haploid cell with a chromosome doubling agent. Also provided are methods for producing transgenic homozygous plants and seeds. The methods comprise transforming a cell from haploid somatic tissue such as embryo, meristem, leaf, root, inflorescence, callus tissue derived from such tissue, or seed and then contacting the transformed cell with a chromosome doubling agent. The methods provide homozygous plant cells which can be regenerated into a plant containing homozygous genes. The methods avoid time consuming crossing methods to obtain a homozygous trait of interest. The methods can be useful for functional genomics, such as knock-out analysis, functional analysis of recessive genes, gene replacement, gene targeting, transgene stacking, and evaluating lethal versus non-lethal analysis of genes. With the current diploid transformation system, these analyses are very complicated and costly. The inventive methods can be used to transform and express recessive genes in T0 plants. [0010]
  • Haploid induction systems have been developed for various plants to produce haploid tissues, plants and seeds. The haploid induction system can produce haploid plants from any genotype by crossing a selected line (as female) with an inducer line. Such inducer lines for maize include Stock 6 (Coe, 1959, [0011] Am. Nat. 93:381-382; Sharkar and Coe, 1966, Genetics 54:453-464) RWS (Roeber and Geiger 2001, submitted to Crop Science), KEMS (Deimling, Roeber, and Geiger, 1997, Vortr. Pflanzenzuchtg 38:203-224), or KMS and ZMS (Chalyk, Bylich & Chebotar, 1994, MNL 68:47; Chalyk & Chebotar, 2000, Plant Breeding 119:363-364), and indeterminate gametophyte (ig) mutation (Kermicle 1969 Science 166:1422-1424). The disclosures of which are incorporated herein by reference.
  • Methods for obtaining haploid plants are also disclosed in Kobayashi, M. et al., [0012] Journ. of Heredity 7(1):9-14, 1980, Pollacsek, M., Agronomie (Paris) 12(3):247-251,1992; Cho-Un-Haing et al., Journ. of Plant Biol., 1996, 39(3):185-188; Verdoodt, L., et al., February 1998, 96(2):294-300; Genetic Manipulation in Plant Breeding, Proceedings International Symposium Organized by EUCARPIA, Sep. 8-13, 1985, Berlin, Germany; Chalyk et al., 1994, Maize Genet Coop. Newsletter 68:47; Chalyk, S. T., 1999, Maize Genet. Coop. Newsletter 73:53-54; Coe, R. H., 1959, Am. Nat. 93:381-382; Deimling, S. et al., 1997, Vortr. Pflanzenzuchtg 38:203-204; Kato, A., 1999, J. Hered. 90:276-280; Lashermes, P. et al., 1988, Theor. Appl. Genet. 76:570-572 and 76:405-410; Tyrnov, V. S. et al., 1984, Dokl. Akad. Nauk. SSSR 276:735-738; Zabirova, E. R. et al., 1996, Kukuruza I Sorgo N4, 17-19; Aman, M. A., 1978, Indian J. Genet Plant Breed 38:452-457; Chalyk S.T., 1994, Euphytica 79:13-18; Chase, S. S., 1952, Agron. J. 44:263-267; Coe, E. H., 1959, Am. Nat. 93:381-382; Coe, E. H., and Sarkar, K. R., 1964 J. Hered. 55:231-233; Greenblatt, I. M. and Bock, M., 1967, J. Hered. 58:9-13; Kato, A., 1990, Maize Genet. Coop. Newsletter 65:109-110; Kato, A., 1997, Sex. Plant Reprod. 10:96-100; Nanda, D. K. and Chase, S. S., 1966, Crop Sci. 6:213-215; Sarkar, K. R. and Coe, E. H., 1966, Genetics 54:453-464; Sarkar, K. R. and Coe, E. H., 1971, Crop Sci. 11:543-544; Sarkar, K. R. and Sachan J. K. S., 1972, Indian J. Agric. Sci. 42:781-786; Kermicle J. L., 1969, Mehta Yeshwant, M. R., Genetics and Molecular Biology, September 2000, 23(3):617-622; Tahir, M. S. et al. Pakistan Journal of Scientific and Industrial Research, August 2000, 43(4):258-261; Knox, R. E. et al. Plant Breeding, August 2000, 119(4):289-298; and U.S. Pat. No. 5,639,951 the disclosures of which are incorporate herein by reference.
  • Somatic haploid cells, haploid embryos, haploid seeds, or haploid seedlings produced from haploid seeds can be treated with a chromosome doubling agent. Homozygous plants can be regenerated from haploid cells by contacting the haploid cells, such as embryo cells or callus produced from such cells, with chromosome doubling agents, such as coichicine, anti-microtubule herbicides, or nitrous oxide to create homozygous doubled haploid cells. Treatment of a haploid seed or the resulting seedling generally produces a chimeric plant, partially haploid and partially doubled haploid. It may be beneficial to nick the seedling before treatment with colchicine. When reproductive tissue contains doubled haploid cells, then doubled haploid seed is produced. [0013]
  • Haploid embryos, haploid seeds, or somatic haploid cells from a haploid plant can be harvested and transformed by any known means. Transgenic homozygous plants can be regenerated from the transformed cells as described above. Transgenic homozygous seeds can also be produced by the method described above by treating a haploid seed or the resulting seedling with a chromosome doubling agent and growing the seed to produce a plant having homozygous seeds. [0014]
  • Methods of chromosome doubling are disclosed in Antoine-Michard, S. et al., Plant cell, tissue organ cult., Cordrecht, the Netherlands, Kluwer Academic Publishers, 1997, 48(3):203-207; Kato, A., Maize Genetics Cooperation Newsletter 1997, 36-37; and Wan, Y. et al., TAG, 1989, 77: 889-892. Wan, Y. et al., TAG, 1991, 81: 205-211. The disclosures of which are incorporated herein by reference. Typical methods involve contacting the transformed cell with nitrous oxide, anti-microtubule herbicides, or coichicine. [0015]
  • Polynucleotides or polypeptides involved in growth stimulation or cell cycle stimulation can be used to increase the frequency of haploid embryos produced per ear, increase the recovery of transformed haploid plants, and/or stimulate chromosomal doubling efficiency. The growth stimulation polynucleotide can be provided by either the female or male parent. The growth stimulation polynucleotide or polypeptide can be provided by stable or transient transformation. [0016]
  • Polynucleotides whose overexpression has been shown to stimulate the cell cycle include Cyclin A, Cyclin B, Cyclin C, Cyclin D, Cyclin E, Cyclin F, Cyclin G, and Cyclin H; Pin1; E2F; Cdc25; RepA and similar plant viral polynucleotides encoding replication-associated proteins. In addition, there are other cell cycle regulatory polynucleotides whose expression must be down-regulated to stimulate the cycle and concomitant cell division. These include polynucleotides whose encoded polypeptides normally repress the cell cycle, such as Rb, CKI, prohibitin, and wee1. Thus, polynucleotides that encode polypeptides involved in the regulation of the cell cycle in plants can be used in the invention, and include cyclins (Doerner (1994) Plant Physiol. 106:823-827.), maize cdc2 (Colasanti et al. (1991) PNAS 88:3377-3381), other cdc2 WO 99/53069, cdc25+ (Russell and Nurse (1986) Cell 45:145-153), the geminivirus RepA gene (U.S. Ser. No. 09/257,131), plant E2F (Ramirez-Parra et al. (1999) Nuc. Ac. Res. 27:3527-3533 and Sekine et al. (1999) FEBS Left. 460:117-122), Pin1 (Liou et al., 2002, Proc Natl Acad Sci U S A 99(3):1335-40 and Yao et al., 2001, J Biol Chem 276(17):13517-23), Cyclin D disclosed in WO 00/17364 published Mar. 30, 2000, CKS polynucleotides disclosed in 99/61619 filed May 19, 1999, Cyclin E polynucleotides disclosed in 09/496,444 filed Feb. 2, 2000. Repressors of the cell cycle such as Rb (Grafi et al. (1996) Proc Natl Acad Sci 93(17): 8962-7; Ach et al. (1997) Mol Cell Biol 17(9):5077-86), CKI (US 01/44038 filed Nov. 6, 2001), prohibitin (WO 00/15818), and wee1 (disclosed in WO 00/37645) genes can be used in the practice of the invention. The disclosures of which are herein incorporated by reference. [0017]
  • Examples of plant virus replicase polynucleotide sources suitable for growth stimulation (i.e. stimulation of S-phase in the cell cycle) include wheat dwarf virus, maize streak virus, tobacco yellow dwarf virus, tomato golden mosaic virus, abutilon mosaic virus, cassaya mosaic virus, beet curly top virus, bean dwarf mosaic virus, bean golden mosaic virus, chloris striate mosaic virus, digitaria streak virus, miscanthus streak virus, maize streak virus, panicum streak virus, potato yellow mosaic virus, squash leaf curl virus, sugarcane streak virus, tomato golden mosaic virus, tomato leaf curl virus, tomato mottle virus, tobacco yellow dwarf virus, tomato yellow leaf curl virus, African cassaya mosaic virus, and the bean yellow dwarf virus. Replicase from the wheat dwarf virus has been sequenced and functionally characterized. Replicase binds to a well-characterized binding motif on the Rb protein (Xie et al., [0018] The EMBO Journal Vol. 14 no.16 pp. 4073-4082, 1995; Orozco et al., Journal of Biological Chemistry, Vol.272, No. 15, pp. 9840-9846,1997; Timmermans et al., Annual Review Plant Physiology. Plant Mol. Biol, 45:79-112,1994; Stanley, Genetics and Development 3:91-96,1996; Davies et al., Geminivirus Genomes, Chapter 2, and Gutierrez, Plant Biology 1:492-497,1998). Other growth stimulation (S-phase stimulating) polynucleotides suitable for use include viral cell cycle modulator proteins such as CLINK (Aronson et al Journal of Virology 74:2968-2972, 2000). Examples of other viral sources for this type of protein include banana bunchy top virus, milk vetch dwarf virus, subterranean clover stunt virus Ageratum yellow vein virus and other representatives of plant nanoviruses. The disclosures of these items are incorporated herein by reference.
  • Growth stimulation polynucleotides include polynucleotides whose overexpression stimulates growth through triggering developmental programs include such examples as SERK, Lec1, Lec2, WUS, FUS3, ABI3 (Vp1), BMN3, ANT, and members of the Knotted family, such as Kn1, STM, OSH1, and SbH1; cytokinin genes such as IPT, TZS, CKI-1; and genes that produce growth stimulating peptides such as PSK. Also, genes whose encoded products repress specific plant developmental programs can be down-regulated to stimulate growth, such as the gene PICKLE, that when down-regulated results in embryogenic growth. Thus, these genes useful in the present invention include the Kn1 family of genes disclosed in Vollbrecht et al., [0019] Nature 350:241-243,1991; Sentoku et al., Develop. Biol. 220:358-364, 2000 and Sinha et al., 1993, Genes Dev 7(5):787-95, WUSCHEL or WUS genes found in Mayer et al., Cell 95:805-815,1998; Lenhard et al., Cell 105(6):805-14; and Laux et al. 1996, Development 122(1):87-96, Lec1 polynucleotides disclosed in US99/26514 filed Nov. 9, 1999, SERK polynucleotides disclosed in Schmidt et al. 1997, Development 124(10):2049-62 and Baudino et al. 2001, Planta 213(1):1-10, Babyboom (BMN3) polynucleotides disclosed in EP1 057891 (A1), LEC2 polynucleotides disclosed in Stone et al. 2001, Proc Natl Acad Sci U S A 98(20): 11806-11, FUS3 polynucleotides disclosed in Nambara et al. 2000, Dev Biol 220(2):412-23 and Vicient et al., 2000, J Exp Bot 51(347):995-1003, STM polynucleotides disclosed in Endrizzi et al. 1996, Plant J 10(6):967-79 and Long et al. 1996, Nature 379(6560):66-9. 8, ANT (Aintegument) polynucleotides disclosed in Mizukami, 2001, Curr Opin Plant Biol 4(6):533-9; Nole-Wilson S, Krizek BA., 2000, Nucleic Acids Res 28(21):4076-82; Mizukami Y, Fischer R L., 2000, Proc Natl Acad Sci U S A 97(2):942-7; and Krizek B A., 1999, Dev Genet 25(3):224-36, ABI3 polynucleotides disclosed in Suzuki et al. 2001, Plant J 28(4):409-18; Rohde et al., 2000, Trends Plant Sci 5(10):418-9; Parcy et al., 1997, Plant Cell 9(8):1265-77; Parcy F, Giraudat J., 1997, Plant J 11 (4):693-702, and PICKLE (Ogas et al., PNAS 96:13839-13844,1999). Genes that stimulate growth by encoding products involved in the synthesis of growth stimulating hormones (IPT, TZS), that confer independence from a hormone (CKI1) or in which the peptide itself is a growth stimulating hormone (PSK). Thus, such genes can be used in the present invention and include the IPT gene of Agrobacterium tumefaciens (Strabala et al. (1989) Mol. Gen. Genet. 216:388-394, Bonnard et al. (1989) Mol Gen. Genet. 216:428-438, DDBJ/EMBL/GenBank), TZS (Beaty et al. (1986) Mol. Gen. Genet. 203:274-280, Akiyoshi et al. (1985) Nucleic Acids Res. 13:2773-2788, Regier et al. (1989) Nucleic Acids Res. 17:8885), CKI1 (Kakimoto (1996) Science 274:982-985), and PSKα (Yang et al. (1999) PNAS 96:13560-13565). The disclosures of the above are incorporated herein by reference.
  • As discussed above, growth stimulation polynucleotides (or polypeptides) can be used to increase chromosomal doubling in haploid plant tissues (callus, seeds, seedlings etc.) with the methods described herein. The frequency of doubled haploids can be increased several fold. The growth stimulation polynucleotides can be introduced into the male or female parent. Introducing the growth stimulation polynucleotides into the maternal parent will result in a plant homozygous for the growth stimulation polynucleotide. If the growth stimulation polynucleotide is introduced into the paternal parent (the inducer line) the growth stimulation polynucleotide would be present in the endosperm, but not in the embryo. This can result in increased vigor of the haploid embryo. [0020]
  • After successful doubling of the haploid chromosomes, it may be desirable to remove the above growth stimulation polynucleotides. This can be accomplished by using various methods of gene excision, such as those described below including the use of recombination sites and recombinases. [0021]
  • In another aspect the inducer line may contain a scorable marker gene, for example colored markers in the endosperm, embryo or stem. Such markers include GUS (U.S. Pat. Nos. 5,599,670 and 5,432,081), GFP (U.S. Pat. Nos. 6,146,826; 5,491,084; and WO 97/41228), luciferase (U.S. Pat. No. 5,674,713 and Ow et al. 1986 [0022] Science 234 (4778) 856-859), CRC (Ludwig et al., 1990) other anthocyanin genes such as A, C, R-nj, etc. and others known in the art. The disclosures of which are incorporated herein by reference. When the inducer line is crossed with the selected line, the resulting haploid seeds will have colored endosperm with colorless embryo. Some lines already contain a color marker. For various reasons it may be desirable to express the marker gene in the embryo. In particular, it may be desirable to express the marker gene in the early stage of development, about 8-15 days after pollination using an appropriate promoter such as an oleosin or a Lec1 promoter. Marker negative embryos are then selected to obtain haploid embryos. This method provides the advantage of obtaining haploid embryos without marker genes.
  • The methods of the invention can be practiced with any plant. Such plants include but are not limited to maize, soybean, oilseed Brassica, alfalfa, rice, rye, sorghum, sunflower, tobacco, potato, peanuts, cotton, sweet potato, cassaya, sugar beets, tomato, oats, barley, and wheat. [0023]
  • Genes of interest are reflective of the commercial markets and interests of those involved in the development of the crop. Crops and markets of interest change, and as developing nations open up world markets, new crops and technologies will emerge also. In addition, as our understanding of agronomic traits and characteristics such as yield and heterosis increases, the choice of genes for transformation will change accordingly. It is also understood that two or more genes may be introduced into a plant. [0024]
  • General categories of genes of interest include for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding agronomic traits, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Genes of interest also include those involved in oil, starch, carbohydrate, or nutrient metabolism as well as those affecting for example kernel size, sucrose loading, and the like. The quality of grain is reflected in traits such as levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and levels of cellulose. [0025]
  • Grain traits such as oil, starch, and protein content can be genetically altered. Modifications include increasing the content of oleic acid, saturated or unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and also modification of starch. Hordothionin protein modifications are described in U.S. Pat. No. 5,990,389 issued Nov. 23,1999, U.S. Pat. No. 5,885,801 issued Mar. 23, 1999, U.S. Pat. No. 5,885,802 issued Mar. 23, 1999 and U.S. Pat. No. 5,703,409. Another example is lysine and/or sulfur rich seed protein encoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016 issued Dec. 15, 1998, and the chymotrypsin inhibitor from barley, Williamson et al. (1987) [0026] Eur. J. Biochem. 165:99-106. The disclosures of the above are herein incorporated by reference.
  • Derivatives of the coding sequences can be made by site-directed mutagenesis to increase the level of preselected amino acids in the encoded polypeptide. For example, the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor WO98/20133 the disclosure of which is incorporated herein by reference. Other proteins include methionine-rich plant proteins such as from corn (Pedersen et al. (1986) [0027] J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; and rice (Musumura et al. (1989) Plant Mol. Biol. 12:123). The disclosures of which are incorporated herein by reference. Other genes encode latex, Floury 2, growth factors, seed storage factors, and transcription factors.
  • Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like. Such genes include, for example [0028] Bacillus thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene 48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825); and the like. Genes encoding disease resistance traits include detoxification genes, such as against fumonisin (U.S. Pat. No. 5,792,931, issued Aug. 11, 1998); avirulence (avr) and disease resistance genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089); and the like.
  • Herbicide resistance traits may include genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance, in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene encodes resistance to the herbicide chlorsulfuron. Glyphosate tolerance can be obtained from the EPSPS gene. [0029]
  • Sterility genes can also be encoded in an expression cassette and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development. [0030]
  • Commercial traits can also be encoded on a gene or genes that could increase for example, starch for ethanol production, or provide expression of proteins. Another commercial use of transformed plants is the production of polymers and bioplastics such as described in U.S. Pat. No. 5,602,321 issued Feb. 11, 1997. Genes such as B-Ketothiolase, PHBase (polyhydroxybutyrate synthase) and acetoacetyl-CoA reductase (see Schubert et al. (1988) [0031] J. Bacteriol. 170:5837-5847) facilitate expression of polyhyroxyalkanoates (PHAs). Genes of medicinal and pharmaceutical uses, such as that encoding avidin and vaccines or proteins produced utilizing plants as factories are also contemplated as part of this invention.
  • It is recognized that the present invention contemplates the use of various gene targeting methods. Insertion, excision or recombination sites for use in the invention are known in the art and include FRT or lox sites (see, for example, Schlake et al. (1994) [0032] Biochemistry 33:12746-12751; Huang et al. (1991) Nucleic Acids Res. 19:443-448; Sadowski (1995) Prog. Nuc. Acid Res. Mol. Bio. 51:53-91; Cox (1989) Mobile DNA, ed. Berg and Howe (American Society of Microbiology, Washington D.C.), pp.116-670; Dixon et al. (1995) 18:449-458; Umlauf et al. (1988) EMBO J. 7:1845-1852; Buchholz et al. (1996) Nucleic Acids Res. 24:3118-3119; Kilby et al. (1993) Trends Genet. 9:413-421; Roseanne et al. (1995) Nat. Med. 1:592-594; Albert et al. (1995) Plant J. 7:649-659; Bailey et al. (1992) Plant Mol. Biol. 18:353-361; Odell et al. (1990) Mol. Gen. Genet. 223:369-378; and Dale et al. (1991) Proc. Natl. Acad. Sci. USA 88:10558-105620; lox (Albert et al. (1995) Plant J. 7:649-659; Qui et al. (1994) Proc. Natl. Acad. Sci. USA 91:1706-1710; Stuurman et al. (1996) Plant Mol. Biol. 32:901-913; Odell et al. (1990) Mol. Gen. Genet 223:369-378; Dale et al. (1990) Gene, 91:79-85; and Bayley et al. (1992) Plant Mol. Biol. 18:353-361); U.S. Pat. No. 5,658,772; U.S. Pat. No. 4,959,317; U.S. Pat. No. 6,110,736. Such recombination sites in the presence of a compatible recombinase allow for the targeted integration of one or more nucleotide sequences of interest into the plant genome. It is recognized that variations of targeted insertion can also be practiced with the invention. See for example WO 99/25821; WO 99/25855; WO 99/25840; WO 99/25853. The disclosures of the above are herein incorporated by reference.
  • Where appropriate, the nucleotide sequences of interest may be optimized for increased expression in the plant. Where mammalian, yeast, or bacterial genes are used in the invention, they can be synthesized using plant-preferred codons for improved expression. It is recognized that for expression in monocots, dicot genes can also be synthesized using monocot-preferred codons. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, 5,436, 391, and Murray et al. (1989) [0033] Nucleic Acids Res. 17:477-498, herein incorporated by reference.
  • The plant-preferred codons may be determined from the codons utilized more frequently in the proteins expressed in the recipient plant of interest. It is recognized that monocot-or dicot-preferred sequences may be constructed as well as plant-preferred sequences for particular plant species. See, for example, EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al. (1991) [0034] Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Res. 17:477-498; U.S. Pat. Nos. 5,380,831 and 5,436,391; and the like, herein incorporated by reference. It is further recognized that all or any part of the gene sequence may be optimized or synthetic. That is, fully optimized or partially optimized sequences may also be used.
  • Additional sequence modifications are known to enhance gene expression in a cellular host and can be used in the invention. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences, which may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence may be modified to avoid predicted hairpin secondary mRNA structures. [0035]
  • In one example, where a DNA construct comprising a compatible recombinase gene is to be used for targeted integration of a nucleotide sequence of interest into a target site within a chromosome of interest, the nucleotide sequence encoding the compatible recombinase may be constructed with plant-preferred codons. More particularly, where the gene encodes a FLP recombinase, for example, the FLP gene sequence may be constructed using plant-preferred codons to obtain an FLP recombinase that is optimized for expression in the plant, WO 99/27077, the disclosure of which is incorporated herein by reference. [0036]
  • The nucleotide sequences of interest may be utilized in an expression cassette. Generally the nucleotide sequence of interest is operably linked with a functional promoter, and in most instances a termination region. There are various ways to achieve the expression cassette within the practice of the invention. In one embodiment of the invention, the nucleotide sequence of interest is transferred or inserted into the genome as an expression cassette. Alternatively, the nucleotide sequence may be inserted into a site within the genome that is 3′ to a promoter region. In this latter instance, the insertion of the coding sequence 3′ to the promoter region is such that a functional expression cassette is achieved upon integration. [0037]
  • For convenience, the nucleotide sequences of interest are generally provided in expression cassettes for expression in the plant. The cassette will include 5′ and 3′ regulatory sequences operably linked to a nucleotide sequence of interest. By “operably linked” is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the nucleic acid sequence corresponding to the second sequence. The cassette may additionally contain at least one additional gene or nucleotide sequence of interest to be cotransformed into the plant. Thus, each nucleic acid sequence will be operably linked to 5′ and 3′ regulatory sequences. Alternatively, the additional gene(s) or nucleotide sequence(s) can be provided on multiple expression cassettes. [0038]
  • The construction of such expression cassettes which can be employed in conjunction with the present invention is well known to those of skill in the art in light of the present disclosure. See, e.g., Sambrook et al., [0039] Molecular Cloning: A Laboratory Manual; Cold Spring Harbor, N.Y.; (1989); Gelvin et al., Plant Molecular Biology Manual (1990); Plant Biotechnology: Commercial Prospects and Problems, eds. Prakash et al., Oxford & IBH Publishing Co.; New Delhi, India; (1993); and Heslot et al., Molecular Biology and Genetic Engineering of Yeasts; CRC Press, Inc., USA; (1992); each disclosure incorporated herein by reference.
  • For example, plant expression vectors may include (1) a cloned plant gene under the transcriptional control of 5′ and 3′ regulatory sequences and (2) a dominant selectable marker. Such plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal. Such an expression cassette is generally provided with a plurality of restriction sites for insertion of the nucleotide sequence of interest that is to be under the transcriptional regulation of the regulatory regions. [0040]
  • The expression cassette may additionally contain selectable marker genes. The marker gene confers a selectable phenotype on plant cells. Usually, the selectable marker gene will encode antibiotic or herbicide resistance. Suitable genes include those coding for resistance to the antibiotics spectinomycin and streptomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance. [0041]
  • Suitable genes coding for resistance to herbicides include those which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), those which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta and the ALS gene encodes resistance to the herbicide chlorsulfuron. [0042]
  • Selectable marker genes for the selection of transformed cells or tissues are disclosed in the following publications. See generally, Yarranton (1992) [0043] Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol 6:2419-2422; Barkley et al. (1980) Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; M. Gossen (1993) Ph.D dissertation, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell Bio. 10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) Topics in Mol. and Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Gatz et al. (1992) Plant J. 2:397-404; A. L. Bonin (1993) Ph.D. dissertation, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Exp. Pharmacology 78; Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference.
  • The expression cassette will generally include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region, a nucleotide sequence of interest, and a transcriptional and translational termination region functional in plants. The transcriptional initiation region, the promoter, may be native or analogous or foreign or heterologous to the plant host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By “foreign” is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced. [0044]
  • While it may be preferable to express the nucleotide sequences of interest using heterologous promoters, the native promoter sequences may be used. Such constructs would change expression levels of any protein encoded by a nucleotide sequence of interest in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered. [0045]
  • Constitutive, tissue-preferred or inducible promoters can be employed. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′-promoter derived from T-DNA of [0046] Agrobacterium tumefaciens, the actin promoter, the ubiquitin promoter, the histone H2B promoter (Nakayama et al., 1992, FEBS Lett 30:167-170), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter, and other transcription initiation regions from various plant genes known in the art.
  • Examples of inducible promoters are the Adh1 promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, the PPDK promoter which is inducible by light, the In2 promoter which is safener induced, the ERE promoter which is estrogen induced and the Pepcarboxylase promoter which is light induced. [0047]
  • Examples of promoters under developmental control include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers. An exemplary promoter is the anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and 5,689,051). Examples of seed-preferred promoters include, but are not limited to, 27 kD gamma zein promoter and waxy promoter, Boronat, A., Martinez, M. C., Reina, M., Puigdomenech, P. and Palau, J.; Isolation and sequencing of a 28 kD glutelin-2 gene from maize: Common elements in the 5′ flanking regions among zein and glutelin genes; [0048] Plant Sci. 47:95-102 (1986) and Reina, M., Ponte, I., Guillen, P., Boronat, A. and Palau, J., Sequence analysis of a genomic clone encoding a Zc2 protein from Zea mays W64 A, Nucleic Acids Res. 18(21):6426 (1990). See the following site relating to the waxy promoter: Kloesgen, R. B., Gierl, A., Schwarz-Sommer, Z. S. and Saedler, H., Molecular analysis of the waxy locus of Zea mays, Mol. Gen. Genet. 203:237-244 (1986). The disclosures of each of these are incorporated herein by reference. The barley or maize Nuc1 promoter, the maize Cim 1 promoter or the maize LTP2 promoter can be used to preferentially express in the nucellus. See for example U.S. Serial No. 60/097,233 filed Aug. 20, 1998 the disclosure of which is incorporated herein by reference.
  • Either heterologous or non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention. These promoters can also be used, for example, in expression cassettes to drive expression of antisense nucleic acids to reduce, increase, or alter concentration and/or composition of the proteins of the present invention in a desired tissue. [0049]
  • The termination region is optional and may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the potato proteinase inhibitor (PinII) gene o