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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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
• • 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
  • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
• • 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)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis

Definitions

• This invention involves a method for visually identifying and selecting trangenic plant cells or tissues by carotenoid pigmentation.
• Antibiotics such as kanamycin, G418, hygromycin, bleomycin and streptomycin, among others, have been used for the selection of transgenic plants (Bevan et al . , (1983), Nature 394:184-187; Dekeyser et al. , (1989), Plant Phvsiol. 90:217-223; Hille et al., (1986), Plant Mol. Biol. 7:171-176; Jones et al., (1987), Mol. Gen. Gen. 210: 86-91; Mulsant et al., (1988), Som. Cell Mol. Gen. 14:243-252; Van den Elzen et al . , (1985), Plant Mol. Biol. 5:299-302; Waldron et al . , (1985), Plant Mol. Biol. 5:103-108) .
• kanamycin which is used commonly, is not effective as a selective agent m gramineaceous plants; some plants from this group car. tolerate up to 800 mg/L of kanamycin (Dekeyser et ai., (1989), Plant Phvsiol. 90:217-223; Haupt ann et al . , (1988), Plant Phvsiol. 86:602-606) .
• Herbicides such as chlorsulfuron, 2,4-D, glyphosate, phosphmotricin, and others, have been proposed as selective agents (DeBlock et al., (1987), EMBO J. 6:2513- 2518; Dekeyser et al . , (1989), Plant Phvsiol. 90:217-223; Li et al., (1992), Plant Phvsiol. 100:662-668; Streber, W.R.,and ill itzer, L., (1989), Bio/Technolo ⁇ v 7:811; Shah et al., (1986), Science 233: 478-481; White et al . , (1990), Nucl.
• ammo acids such as lysine and threonine, or the lysine derivative amino ethyl cysteine, can also be used as selective agents due to their ability to inhibit cell growth when applied at h gh concentrations (Shaul 0. and Galili G., (1992), Plant J. 2:203-209; Perl et al . , (1993), Bio/Technolo ⁇ y 11:715-718) .
• expression of the selectable marker gene which allows the transgenic cells to grow under selection, results in overproduction of these ammo acids by transgenic cells. In some cases, this results abnormal plant development (Shaul 0. and Galili G., (1992), Plant J. 2:203-209) .
• Visual markers reduce tne use of toxic additives the regeneration medium. They also allow for a non-destructive, real-time assessment of transformation frequency and the dynamics of transgenic tissue regeneration (Casas et al . , (1993), PNAS 90:11212-11216; Yoder et al . , (1994), Euohvtica 79:163- 167) .
• Visual markers based on the expression of anthocyanin, a natural plant pigment have been proposed for use m the identification of transgenic cell lineages in both tomato and corn (Yoder et al . , (1994), Euohvtica 79:163-167; Pioneer Hi-Bred International, patent application WO91/02059) .
• Carotenoids are a ubiquitous group of molecules that are found many organisms. In plants, carotenoids protect cells and tissues aga st the deleterious effects caused by photosensitized oxidation and are used as accessory pigments light harvesting. In mammals, carotenoids are precursors of vitamin A and are now receiving attention as one of the nutritional factors with potential anti-cancer activity. Carotenoids are also produced by some types of bacteria.
• Phytoene is the first carotenoid the biosynthesis pathway and is produced by the dimerization of a 20-carbon atom precursor, geranylgeranyl pyrophosphate (GGPP) .
• GGPP geranylgeranyl pyrophosphate
• the red carotenoid lycopene is the next carotenoid, and is produced from the phytoene in the pathway.
• Lycopene is biosynthetically synthesized from phytoene through four sequential dehydrogenation reactions by the removal of eight atoms of hydrogen.
• a dehydrogenase enzyme catalyzes the conversion of phytoene mto lycopene. This enzyme removes four moles of hydrogen from each mole of phytoene, and is referred to as phytoene dehydrogenase-4H.
• Beta-carotene is the next carotenoid produced the carotenoid biosynthesis pathway. Beta-carotene is produced by the cyclization of unsaturated carotenoi ⁇ s m a procedure not yet well understood (Bramley et al, (1988) In: Current Topics m Cellular Regulation 29:291,297) . It is believed that in both plants and microorganisms a smgle cyclase is responsible for the conversion of lycopene to Beta-carotene.
• Zeaxanthin and zeaxanthin diglucoside are the fourth and fifth carotenoids produced in the Erwinia genus carotenoid biosynthesis pathway. These carotenoids are useful as a food colorants, and are used as colorants in the poultry industry.
• WO91/13078 describes the characterization and expression of six genes from the Gram-negative bacteria Erwinia herhi cola . These genes encode the enzymes geranylgeranyl pyrophosphate (GGPP) synthase, phytoene synthase, phytoene dehydrogenase - 4H, lycopene cyclase, Beta-carotene hydroxylase and zeaxanthin glycosylase.
• GGPP geranylgeranyl pyrophosphate
• European patent application 393690 describes the characterization and expression of six genes from the gram-negative bacteria Erwini a uredovora .
• the present invention involves a method for visually identifying and subsequently regenerating transgenic plants.
• the present invention also provides a method for the visual identification of proprietary transgenic germplasm.
• the method for visually identifying transgenic plant cells or tissues involves culturing non-transgenic (or non- transformed) and transgenic plant cells m a culture medium.
• the transgenic plant cells or tissues contam a heterologous, recombinant chimeric DNA segment which contains at least one expression cassette.
• An example of the plant cells or tissues that can be used this method include but are not limited to tomato, cucurbits, pepper, lettuce and carrots.
• At least one expression cassette must contain a promoter DNA segment which functions in specific plant cells to cause the production of an RNA sequence from the DNA segment described as the second component.
• the second component is a DNA segment which contains a plastid targeting signal fused to the ammo-terminal end of the coding region of the phytoene synthase gene from the Erwinia group of genes, which when expressed results m the production of a carotenoid.
• the preferred gene is the phytoene synthase gene from Erwinia herbicola .
• the DNA segment contammg the plastid targeting signal and phytoene synthase gene produces mRNA which encodes a chimeric polypeptide.
• the chimeric polypeptide is produced m the cytoplasm and then transported to the plastids of the plant cells by the plastid targeting signal contained the DNA segment.
• the third component of the expression cassette is a 3' non-translated DNA segment.
• This segment contains sequences that in plant cells or tissues result in the termination of transcription ana additional sequences tnat when transcribed into RNA result in the addition of a polyadenylate tract of residues to tne 3' end of the RNA, which encodes the chimeric polypeptide.
• the transgenic plant cells or tissues may also contain a heterologous, recombinant chimeric DNA segment which contains additional expression cassettes.
• the first expression cassette is the same as the expression cassette described above. It contains a suitable promoter DNA segment, a DNA segment containing a plastid targeting signal fused to the ammo-terminal end of the coding region of the phytoene synthase gene from the Erwinia group of genes which when expressed results m the production of a carotenoid, and a 3' non-translated termination segment.
• the second and subsequent expression cassettes will each contam a promoter segment that controls the expression of a DNA segment which encodes a second gene that is expressed in the transformed plant, and a 3' non- translated segment.
• the second and subsequent genes may be any DNA sequence that one wishes to express in plants.
• the transgenic and non-transgenic plant cells or tissues are grown for a sufficient period of time in culture to allow the transgenic plant cells or tissues to express the phytoene synthase gene, and to accumulate a colored carotenoid product.
• Transgenic plant cells are identified from the non-transgenic plant cells by the appearance of orange or red color due to carotenoid pigmentation. Once the transgenic plant cells or tissues are identified, the transgenic plant cells are recovered and regenerated mto plants.
• the recombinant chimeric DNA segment described above can be inserted mto a vector for use m the method of this invention. Any vector can be used m this invention; however, the preferred vectors are those referred to as binary vectors.
• the DNA of interest can be delivered from the vector plasmid to the plant via Agrobacte ⁇ um-mediazed gene transfer.
• the recombinant chimeric DNA segment can be introduced mto the plant cells or tissues py a variety of other techniques which are well known to those skilled in the art such as eiectroporation, microinjection and micropro ectile bombardment.
• the present invention also encompasses transgenic plants which contam the expression cassettes described above as well as seed generated from said transgenic plants .
• the present invention also involves a method for the visual identification of proprietary transgenic germplasm.
• the method involves culturing an explant (e.g. leaf, cotyledon, root or stem fragments) on a culture medium that promotes formation of callus tissue.
• the proprietary transgenic plants contam a heterologous, recombinant chimeric DNA segment which contains at least one expression cassette.
• An example of the plants that can be used m the method mclude but are not limited to tomato, cucurbits, pepper, lettuce and carrots.
• At least one expression cassette contains a promoter DNA segment which functions specific plant cells or tissues to cause the production of an RNA sequence from the DNA segment described as the second component.
• the second component is a DNA segment which contains a plastid targeting signal fused to the ammo-terminal end of the coding region of the phytoene synthase gene from the Erwini a group of genes, which when expressed results m the production of a carotenoid.
• the preferred gene is the phytoene synthase gene from Erwinia herbi cola .
• the DNA segment contammg the plastid targeting signal and phytoene synthase gene produces RNA which encodes a chimeric polypeptide.
• the chimeric polypeptide s produce ⁇ m the cytoplasm and then transported to the plastids of the plant cells by the plastid targeting signal contained the DNA segment.
• the third component of the expression cassette is a 3' non-translated DNA segment.
• This segment contains sequences that m plant cells result in the termination of transcription and additional sequences that when transcribed mto RNA result in the addition of a polyadenylate tract of residues to the 3' end of the RNA, which encodes the chimeric polypeptide.
• the proprietary transgenic plants may also contain a heterologous, recombinant chimeric DNA segment which contains additional expression cassettes.
• the first expression cassette is the same as the first expression cassette described above. It contains a suitable promoter DNA segment, a DNA segment containing a plastid targeting signal fused to the ammo-termmal end of the coding region of the phytoene synthase gene from the Erwinia group of genes which when expressed results m the production of a carotenoid, and a 3' non-translated termination segment.
• the second and subsequent expression cassettes will each contam a promoter segment that controls the expression of a DNA segment, which encodes a second gene that is expressed m the transformed plant, and a 3' non- translated segment.
• the second and subsequent genes may be any DNA sequence that one wishes to express in plants.
• the explant e.g. leaf, cotyledon, root or stem fragments
• the explant is cultured for a sufficient period of time under conditions that allow for the creation of callus, and for the calli cells to express the phytoene synthase gene, and to accumulate a colored carotenoid product.
• Transgenic plants are identified by tne appearance of an orange to red colored callus.
• the present mvention involves a plasmid designated as pETO203 having American Type Culture Collection accesion number 97282.
• DESCRIPTION OF THE DRAWINGS Figure 1 shows an orange-pigmented tomato callus that was excised from surrounding callus using the pigmentation as guidance. The green “buds” are new meristems that are differentiating from the orange callus.
• Figure 2 shows recallusmg and shoot regeneration from explants derived from transgenic tomato plants expressing the phytoene synthase gene. The green explant produces orange callus from which green shoots are regenerated.
• Figure 3 is a map of the plasmid vector pETO203.
• the present invention uses carotenoid pigmentation of transgenic plant cells or tissues for two applications: (1) the in vi tro visual selection of transgenic plant cells and (2) the visual identification of proprietary transgenic plants.
• the first application involves a method of visually selectmg transgenic plant cells or tissues from non- transgenic plant cells.
• the first step of the method involves culturing non-trangenic and transgenic plant cells or tissues in vi tro .
• plant cells encompasses any material from a plant which has a nucleus and cytoplasm surrounded by a membrane. It mcludes plants grown m a culture medium as cell suspensions, microspores, protoplasts or explants.
• plant tissues encompasses a group of plant cells organized mto a structural and functional unit.
• tne term "transgenic plant” refers to a plant that contains chromosomally integrated foreign or heterologous DNA.
• the transgenic plant cells used this method contain a heterologous, recombinant chimeric DNA segment whicn contains at least one expression cassette.
• the second application involves a method of visually identifying proprietary transgenic plants.
• the first step of the method involves culturing explants derived from the said proprietary plants m vi tro .
• explants encompasses any organ or tissue from a plant that is amenable to in vi tro culture. Explants can be fragments of roots, stems, hypocotyls, cotyledons, leaves, petioles, petals, etc. Proprietary transgenic plants from which the explants are taken contam a heterologous recombinant chimeric DNA segment which contains at least one expression cassette.
• Heterologous refers to an expression cassette that is not present in a non-transformed cell.
• An expression cassette is a DNA segment that contains a gene to be expressed operatively linked to a DNA segment that contains a promoter and to a DNA termination segment, as well as any other regulatory signals needed to affect proper expression and processing of the expression product.
• the expression cassette used in both applications of this mvention contains a chimeric gene composed of a promoter DNA segment which functions m plant cells or tissues, a chimeric DNA segment which comprises a plastid targeting signal fused to the coding region of the phytoene synthase gene from the Erwinia genus group and a 3' non- translated DNA segment.
• non-transgenic and transgenic plant cells or tissues are cultured m a suitable culture medium and allowed to grow for a sufficient period of time to allow the transgenic plant cells or tissues to express the phytoene synthase gene and accumulate a colored carotenoid product.
• the transgenic plant cells or tissues are then identified by their carotenoid pigmentation. Examples of the types of plants that can be used this method include but are not limited to: tomato, cucurbits, pepper, lettuce and carrots. Cucurbits is defmed as the Cucurbitaceae family, which mcludes squash, melon, pumpkin, and cucumber plants.
• Transgenic plants regenerated as a result of this method contain the expression cassette described above.
• the expression of the phytoene synthase gene in these plants and the resultmg plants from it and the orange pigments of the plant cells can be used as a marker in hybrid seed production. Also protoplasts from these plants can be used to detect the protoplast fusion product based on color.
• explants from proprietary transgenic plants are cultured for sufficient period of time to produce callus and to allow the callus cells to express the phytoene synthase gene. If these proprietary transgenic plants contam the expression cassette described above, then the plants can be identified by the carotenoid pigmentation of the callus. This method can be used to monitor the unauthorized use of proprietary germplasm by a competitor. Examples of plants that can be used the method mclude but are not limited to: tomato, cucurbits, pepper, lettuce and carrots.
• the expression cassette used both applications of this invention contains a suitable promoter DNA segment which functions m plant cells or tissues and is operatively linked to the DNA segment.
• the preferred promoter is a promoter that functions durmg defmed stages of plant regeneration m vi tro, such as the E8 promoter from tomato disclosed Deikman J. and Fischer, R.L. , (1988) EMBO J. , 7:3315-3320, and m Deikman et al . , (1992) Plant Physio . 100:2013-2017, nereby incorporated by reference.
• the E8 promoter has been described as active at the onset of ripening and in unripe fruit treated with exogenous ethylene.
• E8 promoter to express specific transgenes m tomato fruit is well documented (International Application No. PCT/US94/03886) .
• the E8 promoter could be successfully used to express both a naturally occurring tomato gene, such as polygalacturonase (Giovannoni et al., (1989) Plant Cell 1:53-63), and a gene that is not part of the tomato chromosome, such as monell (Penarrubia et al . , (1992; Bio/Technolo ⁇ v 10:561-564) m the fruit.
• the E8 promoter was used to drive AdoMetase expression.
• Several transgenic plants were assayed for their ability to synthesize AdoMetase mRNA using a sensitive RNAase protection assay (RPA) (Example 3) .
• RPA RNAase protection assay
• Figures 6 and 7 show the results of an RPA using the fruit from two transgenic plants (ESKN and SESKN) at different stages of fruit ripening. Other tissues from these plants including immature and mature leaves flowers, and stems were negative from the presence of AdoMetase RNA" (p. 15, lines 11-19) .
• the inventors of this mvention have found that the E8 promoter is active m undifferentiated plant callus tissue during defmed stages of m vi tro plant regeneration.
• sequence ID. NO. 1 The DNA sequence of the E8 promoter that can be used m an expression cassette is disclosed Sequence ID. NO. 1.
• Sequence ID. NO. 1 One skilled the art would recognize that all or part of the DNA sequence of this promoter can be used m this invention.
• the portion of the promoter which confers a response to ethylene m both unripe and ripe fruit and which is found at base pairs 1 to 1089, in Sequence ID. NO. 1, can be removed.
• promoters other than the E8 promoter, which are expressed during plant regeneration could be used.
• An example of such a promoter is the wound-mducible AoPR promoter from
• tissue-specific, organ-specific or inducible promoters can be used if the expression cassette is to be placed in proprietary germplasm, for purposes of proprietary identification. Promoters known to be either tissue-specific, organ-specific or inducible by a variety of external stimuli are well known to those skilled in the art. The following are examples of promoters that direct gene expression an organ-specific manner: root cortex- specific TobRD2 (Mendu N. et al., (1995), Plant Phvsiol. 108:48) ; anther-specific (Riggs, D.C. and Horsch, A., (1995) Plant Phvsiol.
• the expression cassette also contains a DNA segment which comprises a plastid targeting signal and a gene whicn results m the production of carotenoid pigmentation.
• Genes that can be used to produce carotenoid pigment accumulation mclude any phytoene synthase gene from the Erwinia genus group of genes which when expressed result the production of a carotenoid.
• the carotenoid genes of Erwinia herbi cola, disclosed in W091/13078, hereby incorporated by reference and the carotenoid genes of Erwinia uredovora disclosed European Patent Application 393690, hereby incorporated by reference, can be used.
• the preferred gene to be used the expression cassette is the phytoene synthase gene from Erwinia herbi cola which encodes the phytoene synthase enzyme.
• the phytoene synthase enzyme catalyzes a reaction to produce phytoene from geranylgeranyl pyrophosphate.
• phytoene is a precursor of the red carotenoid lycopene.
• Lycopene is the carotenoid that gives tomato fruit their red color.
• the DNA sequence of the phytoene synthase gene of Erwinia herbi cola that can be used m this mvention is mcluded as Sequence ID. NO. 2.
• the ammo acid sequence of this gene is included in Sequence ID. NO. 3.
• the cassette contammg the phytoene synthase gene is transcribed, and mRNA is produced in the nucleus.
• the mRNA is then translated mto a chimeric polypeptide (plastid targeting signal/mature phytoene synthase) in the cytoplasm.
• the plastid targeting signal also referred to as a transit peptide
• the phytoene synthase gene encode a chimeric polypeptide which contains a plastid targeting signal.
• the plastid is the center of different enzymatic activities in the plant cell. More particularly, the plastid is the place in the plant cell where the carotenoid pigments develop. Therefore, in order to obtam the carotenoid pigmentation necessary for use in this invention, the phytoene synthase enzyme must reach the plastid.
• genes from the Erwinia genus group of genes which result in the production of a colored member of the carotenoid biosynthetic pathway can be used this invention either alone or m one or more combinations with the phytoene synthase gene. More specifically, the first expression cassette could contam a DNA segment which comprises a plastid targeting signal and the phytoene synthase gene. The second, and subsequent expression cassettes could contam other genes from the carotenoid biosynthetic pathway. The genes that could be used in these additional expression cassettes could be any gene from the Erwinia genus group of genes which when expressed either alone or in the presence of other carotenoid biosynthetic pathway genes result in the production of a colored carotenoid.
• the phytoene dehydrogenase gene from Erwinia herbi col a which catalyzes the conversion cf phytoene mto the red carotenoid lycopene, could be used in combination with the phytoene synthase gene.
• the DNA and ammo acid sequences of a suitable plastid targeting s ⁇ gna_ that can be used the expression cassette are disclosed in Sequence ID. NOS. 4 and 5.
• a suitable plastid targeting signal or equivalent transit peptide could be used. More specifically, the transit peptide could be obtamed from various sources.
• transit peptides of a cytoplasmic precursor of a chloroplast protein or polypeptide as disclosed in EP 0189707 Bl could be used.
• this gene When the E8 promoter is used in this invention to drive the expression of the phytoene synthase gene, this gene is expressed m the callus of the transformed plant cells resultmg the expression of carotenoid pigmentation in the said callus (See Figures 1 and 2) .
• the callus differentiates mto specific tissues or organs, the colored carotenoid product is not visible, presumably because the E8 promoter directs little or no expression of the phytoene synthase gene m these tissues.
• visible manifestations of the E8- phytoene synthase cassette are not observed after the callus phase until the time for the ripening of the fruit.
• the action of the E8- phytoene synthase cassette again becomes visible. Indeed, tomato fruit develop color prematurely as a result of expression of the E8-phytoene synthase gene expression cassette .
• the expression cassette also contains a 3' non- translated termination segment that is operatively linked to the 3' end of the coding region of the phytoene synthase gene.
• the termination segment should have a polyadenylation signal which functions m plants to cause the addition of polyadenylate nucleotides to the 3' end of mRNA.
• Several termination segments useful m plants are well known and can be used herein.
• One example is the 3' nontranslated region of the nopalme synthase gene (NOS-T) , (Fraley et al., (1983) PNAS 80:4803-4807) used herein.
• NOS-T contains a polyadenylation signal.
• the DNA sequence encoding NOS-T is disclosed m Sequence ID. NO. 6.
• Another terminator is the 3 ' -nontranslated region of the pea rbcS- E9 gene, which can also be used (Coruzzi et al . , (1984)
• the second and subsequent expression cassettes will each contam a DNA segment which encodes a gene that is expressed in the transformed plant.
• the second and subsequent expression cassettes also will each contam a suitable promoter DNA segment which drives the expression of the gene, and a 3' non-translated segment.
• the promoter DNA segment and 3' non-translated segment are operably linked to the DNA segment.
• the promoter used in the second expression cassette may be any promoter that controls the expression of the second gene.
• suitable constitutive promoters that can be used mclude the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, the octopme synthase promoter (P-Ocs) and the nopalme synthase promoter (P- Nos) .
• the gene used in the second expression cassette can be any gene desired.
• exemplary genes m clude those listed m Table 1 below, whose transformations mto plants nave been disclosed the patent citations shown in that Table.
• the second expression cassette also contains a 3' non- translatable termination segment that is operatively linked to the 3' end of the second gene.
• the termination segment should have a polyadenylation signal which functions m plants to cause the addition of polyadenylate nucleotides to the 3' end of mRNA. Any termination segment can be used as discussed with the first expression cassette.
• the recombinant chimeric DNA segment can be inserted mto a vector for use in the method of this invention.
• the most efficient vectors for use m this invention are binary vectors.
• Bmary vector plasmids are derived from E. coli and contam small portions of the tumor-inducmg plasmid from Agrobacteri um tumefaci ens .
• Agrobacteri u - mediated gene transfer to introduce DNA into plant cells is well known in the art (Fraley et al., (1985) Bio/Technolo ⁇ v, 3:629; and Rogers et al. , (1987) Meth. Enzvmol. , 153:253-277) .
• the salient feature of the binary plasmid is that after infection by an Agrobacteri um tumefaciens harboring the plasmid a part of the plasmid DNA is mtegrated mto the plant chromosomal DNA.
• the segments that direct this insertion are referred to as the T-DNA right and left border.
• the right and left T-DNA borders can be as small as 25 base pairs length.
• the vector can also contam DNA sequences that encode for kanamycin or other antibiotic resistance to ensure selection of bacterial cells contammg this vector.
• competent E. coli may be transformed with the vector DNA.
• cells are plated onto a medium that contains an antibiotic. E. coli containing the vector which has a gene that confers antibiotic resistance will grow on a medium containing that antibiotic.
• the recombinant chimeric DNA segment can be introduced mto monocotyledonous or dicotyledonous plant cells or tissues using other techniques such as eiectroporation, microprojectile bombardment, and microinjection.
• plant cells or tissues can be transformed with recombinant chimeric DNA segment contammg the gene cassette or cassettes of interest contammg tne visual selection marker gene.
• Plant tissue that is to be used for transformation is prepared by removing it from the seedlings and cutting into parts suitable for transformation with a vector described above.
• the excised plant parts which are called explants
• the excised plant parts are co-cultivated with the bacteria.
• the explants are transferred to a regeneration medium that is supplemented with an appropriate antibiotic, such as carbenicillm, to eliminate the bacteria.
• an appropriate antibiotic such as carbenicillm
• explants are moved to a fresh medium. Approximately one to two weeks after the transfer, orange or red pigmented sectors become visible on the callusmg edges of the explants.
• Transgenic plant cells or tissues can be visually selected using tne method and vectors described above.
• the method of this invention allows for the visual selection of transgenic plant cells or tissuesand regeneration of transgenic plants without incorporating antibiotic or herbicide resistance genes mto the plant genome.
• Toxic additives such as herbicides, ammo acids or ammo acid analogs are not used during plant culture.
• Antibiotics are used only for a brief period to eliminate the Agrobacteri um during the regeneration process.
• transgenic plants containing the phytoene synthase gene develop fruit color earlier and taste different than non-transgenic fruit.
• pETO203 is a bmary vector that contains the 1.1 kilobase pair (kbp) E8 promoter, a DNA segment contammg a plastid targeting signal fused to the coding region of a phytoene synthase gene and a 3' non ⁇ translated region that supplied a transcription termination signal.
• kbp 1.1 kilobase pair
• pETO203 has been deposited with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland, 20852.
• ATCC American Type Culture Collection
• pETO203 was deposited with the ATCC on September 15, 1995, and assigned ATCC Number 97282.
• the E8 promoter was cloned from L . esculentum, variety VFNT Cherry using the polymerase cham reaction (PCR) .
• the template for the PCR reaction was genomic VFNT Cherry DNA, and syntnesis was primed Dy two synthetic oligonucleotides .
• the primers were designed from the published E8 promoter sequences. The sequence of these oligonucleotides, named
• E8 primers #1 and #2 are shown below:
• E8 primer #1 5' NNG AAT TCA TTT TTG ACA TC 3'
• E8 primer #1 anneals to the 5' end of the reported sequence of the E8 promoter. The first two residues are indicated with an N, which represents any nucleotide. Thus, the E8 primer #1 represents a population of primers with varying nucleotide residues in the first and second positions. Following these two variable residues, the next six residues specify the recognition site for the restriction enzyme EcoRI. E8 primer #2 anneals to an area of the tomato genome that surrounds the initiation codon of the E8 gene. Two mismatches occur between the authentic E8 sequence and the E8 primer #2.
• Plant plastids are organelles that perform many functions. Plastids have their own small genome and the capacity to produce some of their own proteins. However, most of the plastid proteins are produced in the cytoplasm, and are encoded for by nuclear genes . The proteins are synthesized with ammo terminal extensions, which direct the precursor protein to the plastid.
• the ammo terminal extension referred to as the targeting signal or plastid targeting signal (PTS )
• PTS plastid targeting signal
• plastids can differentiate into specialized organelles such as chloroplasts and chromoplasts. Alternatively, plastids can also remam m an undifferentiated state, as they do in callus tissue.
• a PTS from the small subunit of the r ⁇ bulose-1, 5-b ⁇ sphosphate carboxylase oxygenase (RUBISCO) gene can be fused to the phytoene synthase gene.
• PCR amplification and subsequent cloning of the RUBISCO PTS can be performed with primers contammg small mismatches to the template that result in the incorporation of restriction sites at the termini of the PTS PCR product.
• primers can be designed to incorporate an Ncol site near the 5' terminus of the PTS, and an SphI site near the 3' terminus of the PTS.
• E. herbicola phytoene synthase gene (crtB) can be cloned by PCR using E. herbi cola DNA as a template and primers designed from the reported sequence.
• the incorporation of small mismatches between the primers and template that create restriction sites near the termini of the PCR product is a cloning strategy that can facilitate the cloning of the PCR amplified crtB gene.
• primers can be designed to incorporate an SphI site near the 5' terminus of the gene, and an SacI site near the 3' terminus of the gene. Cloning of a termination signal
• the termination signal from the nopalme synthase gene can be cloned from the commercially available binary vector pBI121 (Clontech Co., Palo Altc, California) .
• This segment contains sequences that m plant cells result in the termination of transcription and additional sequences that when transcribed mto RNA result in the addition of a polyadenylate tract of residues to the 3' end of the RNA, which encodes the chimeric polypeptide.
• the DNA fragment containing this genetic element can be obtamed by digestion with the restriction enzymes SacI and EcoRI, followed by gel-purification of the approximately 300 base pair fragment.
• Binary vectors are a preferred way of delivermg transgenic gene cassettes into plant chromosomes, via Agrobacteriurn-mediated transformation.
• Plasmid pET0183 is a preferred parent bmary vector which contains a polylinker between the T-DNA borders.
• Alternative binary vectors can be substituted for pET0183.
• other bmary vectors such as pBIN19 (Bevan, M. (1984) Nucl. Acids Res. 12: 8711-8721), pPZPlOO and pPZP200 (Hajdukiewicz et al., (1994) Plant Mol. Biol. 25: 989-994) can be substituted for pET0183 and serve as parent binary vectors.
• a ligation reaction can be prepared and would include the following DNA fragments:
• Plasmid pET0183 (other plasmids that can substituted for pET0183 mclude BIN19, which is commercially available from Clontech Labs, Palo Alto, California, pPZPlOO and pPZP200, which are Doth available from Dr. Pal Maliga at Waksman Institute, Rutgers
• 1.1 kbp E8 promoter Tne PCR-generated 2.2 kbp E8 promoter digested with Xbal and Ncol, and the 1.1 kop fragment gel-purified.
• Targeting signal The PCR-generated PTS DNA digested with Ncol and SphI.
• Phytoene synthase gene The PCR-generated DNA contammg the phytoene synthase gene digested with SphI and SacI .
• NOS-T pBI121 digested with SacI and EcoRI and the approximately 300 base pair fragment gel-purified.
• Tomato seeds were sterilized in 20o Clorox for 20 mmutes, rinsed 3 times m sterile distilled water and placed on Murashigi and Skoog medium (Gibco) solidified with 10 grams of Noble agar (Gibco) in 135 mm PhytaconTM tissue culture vessels (Sigma, St. Louis, MO) . Seeds were germinated for 72 hours at 25°C the dark, then moved to a lighted shelf under approximately 80 micromol-m "2, s _1 PPFD, at 24-26°C.
• Plant tissue used for transformation was prepared by removing cotyledons from " '-day-old seedlings and cutting them mto three parts (proximal, middle ana distal to the growing pomt) .
• the middle and proximal parts were used for co-cultivation with Agro ⁇ acteri u . They were placed abaxial side down on a sterile filter paper overlaying co-cultivation medium R1F supplemented with 16 g/L glucose, and incubated in the dark for 24 hours.
• Bacterial inoculum was prepared by growing A. t umefaci ens, LBA440 : :pETO203, in 25 ml of AB medium (Chilton et al. , (1974) PNAS 71:3672-3676) supplemented with 50 mg/L kanamycin (K) and 25 mg/L streptomycin (St) (AB dislike-, 0St ,) on a shaker at 28°C, 180 rpm, for 24 hours.
• AB medium Cholton et al. , (1974) PNAS 71:3672-3676
• Bacteria were then pelleted by centrifuging at 8000 rpm for 10 minutes in a Beckman J2-21 centrifuge using a JA-20 rotor.
• the inoculum was supplemented with Acetsyringone (3' 5'd ⁇ methoxy- 4 'hydroxy-acetophenone, Sigma, St. Louis, MO) to a final concentration of 600 micromolar.
• the standard regeneration protocol consisted of two-to-four week culture on R 1/2 300 medium followed by four-week- culture on a hormone-free RO 300 medium. Green tomato shoots that differentiated from the calli either on the R 1/2 300 or RO 300 meidu were detached from the surrounding callus and rooted on the RO 300 medium.
• Regenerated transgenic plants displayed three distinctive characteristics: they produced orange-pigmented callus an m vi tro culture; when grown in the field, fruit from transgenic plants developed color earlier than the nontransgenic control fruit; the fruit from the transgenic plants had a distinctly different taste than control fruit from nontransgenic plants.
• T7/E8PS phytoene synthase gene from Erwinia herbicola driven by the E8 promoter
• T7/E8PS Erwinia herbicola driven by the E8 promoter
• nontransgenic control T7 inbred line was surface sterilized in 20 Clorox for 20 mmutes, rinsed 3 times m sterile distilled water and placed on Murashigi and Skoog medium (Gibco) solidified with 10 grams of Noble agar (GIPCO) i 135 mm Phytacon ⁇ tissue culture vessels iSigma, St. Louis, MO) . They were germinated for 72 hours at 25°C the dark, then moved to a lighted shelf under approximately 80 micromolm ⁇ s "1 PPFD, at 24-26°C.
• GIPCO Noble agar
• Cotyledons from seven-day-old seedlings were used for visual identification of proprietary transgenic plants.
• the cotyledons were cut into three parts: proximal, middle and distal to the growing point.
• the middle and proximal parts were placed abaxial side down on R 1/2 regeneration medium and cultured under 80 micromol m "2 s "1 PPFD, 24-26°C, 16 hours photope ⁇ od.
• After two weeks of culture explants were moved to fresh R 1/2 medium.
• About one week after the last transfer orange-pigmented callus became clearly visible on the callusmg edges of explants from the T7/E8PS plants.
• Explants from control T7 plants produced white and/or green callus that is typically observed during m vi tro regeneration of tomato plants.
• ADDRESSEE Greer, Burns & Crain, Ltd.
• B STREET: 233 South Wacker Drive, Suite 8660, Sears Tower
• TTCAATTCAC TTGGTTGTAA AATAAACTTA AACCAATAAC CAAAGANCTA ATAAATCTGA
• GTTTAGTCCA CAAGTTTTAG TGAGAAGTTT TGCTCAAAAT TTTAGGTGAG AAGGTTTGAT
• GTT ATT CGG GCG AAG ACG ACG AGG GTG ACG CCG CGT CCG GCC GGT CTT
• MOLECULE TYPE protein
• Val lie Arg Ala Lys Thr Thr Arg Val Thr Pro Arg Pro Ala Gly Leu 290 295 300 Trp Gin Arg Pro Val * 305 310
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE protein
• GAATTTCCCC GATCGTTCAA ACATTTGGCA ATAAAGTTTC TTAAGATTGA ATCCTGTTGC
• MOLECULE TYPE DNA (genomic)

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