WO1997014807A1

WO1997014807A1 - A method for visually selecting transgenic plant cells or tissues by carotenoid pigmentation

Info

Publication number: WO1997014807A1
Application number: PCT/US1996/004313
Authority: WO
Inventors: Anna Julia Trulson; Carl Joseph Braun, Iii
Original assignee: Seminis Vegetable Seeds, Inc.
Priority date: 1995-10-16
Filing date: 1996-03-29
Publication date: 1997-04-24
Also published as: EP0873413A1; AU5377896A

Abstract

The present invention involves a method of visually identifying and selecting transgenic plants by carotenoid pigmentation. The method of the present invention involves culturing transgenic plant cells or tissues on a culture medium. The transgenic cells or tissues contain a recombinant chimeric DNA segment which contains at least one expression cassette. This cassette contains three components. The first component is a suitable promoter DNA segment which functions in plant cells or tissues. The second component is a DNA segment which contains a plastid targeting signal fused to the coding region of the phytoene synthase gene of the Erwinia group of genes which when expressed result in the production of a colored member of the biosynthetic pathway. The DNA segment causes the production of RNA which encodes a chimeric polypeptide. The third component is a 3' non-translated DNA segment. After the recombinant chimeric DNA segment is inserted into the plant cells or tissues, transgenic plant cells or tissues are identified by the appearance of orange color due to carotenoid pigmentation. The development of the carotenoid pigmentation can be used to separate transgenic plant cells from the non-transgenic plant cells and to regenerate them into plants, or to visually identify proprietary transgenic plants.

Description

A METHOD FOR VISUALLY SELECTING TRANSGENIC PLANT CELLS OR TISSUES BY CAROTENOID PIGMENTATION

FIELD OF THE INVENTION This invention involves a method for visually identifying and selecting trangenic plant cells or tissues by carotenoid pigmentation.

BACKGROUND OF THE INVENTION

Present systems used for the selection of transgenic plant cells involve the utilization of prokaryotic genes conferring resistance to antibiotics, herbicides, amino acids or amino acid analogs added in toxic concentrations to a regeneration medium. Although generally effective in selecting plant cells, these systems may not be effective in some types of plants, or they may result in abnormal phenotypes of the genetically engineered plants. Additionally, the use of these selective agents may have undesirable side effects and thus have raised concern about their environmental safety.

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) .

However, not all plants are equally sensitive to certain antibiotics. For example, 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/Technoloσv 7:811; Shah et al., (1986), Science 233: 478-481; White et al . , (1990), Nucl. Ac d Res. 18:1062) . The use of a herbicide resistance trait in the identification of transgenic plants may result in increased weedmess of transgenic plants because they can become herbicide-resistant weeds m the alternate years of crop rotation. Further, the use of herbicide resistant crops will increase the herbicide load in the environment.

Certain 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/Technoloαy 11:715-718) . In this selection system 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) . The use of visual markers for the identification of transgenic cells is an alternative to using chemically-based selective agents. 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) .

Genes involved carotenoid biosynthesis are good candidates for use as visual markers m the identification of transgenic cells. 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.

Described below is an outline of carotenoid biosynthesis m the Erwinia genus 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) .

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 carotenoiαs 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.

The biosynthetic pathway of the carotenoids has been elucidated based on extensive chemical evidence. The biosynthetic pathway has been described m WO91/13078 and EP 393690 Al, herein incorporated by reference. More specifically, 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. These enzymes catalyze the formation of geranylgeranyl pyrophosphate and the carotenoids phytoene, lycopene, Beta- carotene, zeaxanthin and zeaxanthin diglucoside, where each formed product (through zeaxanthin) is an immediate precursor for the next named product. European patent application 393690 describes the characterization and expression of six genes from the gram-negative bacteria Erwini a uredovora .

SUMMARY OF THE INVENTION 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 Agrobacteπ um-mediazed gene transfer.

In addition, 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 produceα 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. To identify proprietary transgenic germplasm, the explant (e.g. leaf, cotyledon, root or stem fragments) 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.

Finally, 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.

DETAILED DESCRIPTION OF THE INVENTION 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 . As used herein, the term "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. As used herein, the term "plant tissues" encompasses a group of plant cells organized mto a structural and functional unit. Also as used herem, 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 . As used here in the term "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.

In the method for visually selectmg transgenic plant cells or tissues, 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.

In the method for visually identifying proprietary transgenic plants, 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.

The use of the E8 promoter to express specific transgenes m tomato fruit is well documented (International Application No. PCT/US94/03886) . Previously, it has been shown that 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/Technoloαv 10:561-564) m the fruit.

In a few of these prior art publications, researchers investigated the tissue-specific expression pattern of E8 and collectively found that this promoter is very specific its expression pattern. More specifically, researchers have discovered that the E8 promoter drives expression during defmed stages of fruit development.

In the PCT/US94/03886 application, for example, the E8 promoter was used to drive AdoMetase expression. According to the application, "Several transgenic plants were assayed for their ability to synthesize AdoMetase mRNA using a sensitive RNAase protection assay (RPA) (Example 3) . 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.

The DNA sequence of the E8 promoter that can be used m an expression cassette is disclosed 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. For example, 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.

One skilled the art would recognize that 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

Asparagus (Ozcan et al., (1993) Bio/Technoloαv 11:218-221) .

Additionally, one skilled the art would recognize that either 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. 108:117) ; flower petal-specific Gh-2 and Gh-3 (Yamamoto, E. and Allen, R.D., (1995), Plant Phvsiol . 108:135); phosphate starvation-mducible, and root-specific PIG1 (Liu, C, et al. , (1995), Plant Phvsiol. 108:112) . Regulatory elements from these promoters can be adopted for the purpose of expressing the phytoene syntnase gene m an organ-specific manner as a germplasm marker gene.

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. For example, 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. In the carotenoid biosynthesis pathway, 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. However, although this invention will be described with respect to the phytoene synthase gene from Erwinia herbicola, one skilled in the art would recognize that any of the Erwinia genus phytoene synthase genes whose expression would result the production of a colored member of the biosynthetic pathway can be used.

As used m this invention, 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) allows for the chimeric polypeptide to be transported into the plastid.

It is preferred that the phytoene synthase gene encode a chimeric polypeptide which contains a plastid targeting signal. However, when delivered to the proper location withm the plastid, it is not necessary that the mature polypeptide contam the transit peptide. 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. Methods for inserting a foreign protein or polypeptide mto a chloroplast of a plant are disclosed in EP 189707 Bl, hereby incorporated by reference .

Other 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. For example, 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 sιgna_ that can be used the expression cassette are disclosed in Sequence ID. NOS. 4 and 5. One skilled in the art would recognize, however, that another plastid targeting signal or equivalent transit peptide could be used. More specifically, the transit peptide could be obtamed from various sources. For example, transit peptides of a cytoplasmic precursor of a chloroplast protein or polypeptide as disclosed in EP 0189707 Bl could be used.

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) . This illustrates a tissue-specific, or callus-specific, expression pattern of this gene under the control of the E8 promoter. Once 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. In tomato, visible manifestations of the E8- phytoene synthase cassette are not observed after the callus phase until the time for the ripening of the fruit. In these specific-tissue types 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. The 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) EMBO J. 3:1671-1679) .

As stated earlier, more than one expression cassette may be present. 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. Examples of 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. For example, exemplary genes mclude those listed m Table 1 below, whose transformations mto plants nave been disclosed the patent citations shown in that Table.

TABLE 1

Second Gene Product Citation

HMG-CoA Reductase U.S. 5,306,862

Phosphofructokmase U.S. 5,387,756

Waxy Locus of Wheat (antisense) U.S. 5,365,016 ADP-Glucose pyropnosphorylase (antisense; EP 0 368 506 A. EP 0 455 316 A2 WO 92/11382

Potato-alpha-amylase EP 0 470 145 Bl

Sucrose phosphate synthase EP 0 466 995 A2 EP 0 530 978

Granule-bound starch synthase (antisense) WO 92/11376

Tomato vacuolar invertase (antisense) WO 92/14831

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 . The use of Agrobacteri u - mediated gene transfer to introduce DNA into plant cells is well known in the art (Fraley et al., (1985) Bio/Technoloσv, 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.

Recent technological advances m vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in vectors to facilitate construction of vectors capable of expressing various genes. The vectors described by Hajdukiewicz et al., (1994) Plant Mol. Biol. 25:989-994, which have convenient multi-linker regions can be used in this invention.

One skilled m the art would recognize that if a plasmid vector is used the vector can also contam DNA sequences that encode for kanamycin or other antibiotic resistance to ensure selection of bacterial cells contammg this vector. After the vector DNA is prepared, competent E. coli may be transformed with the vector DNA. In order to select E. coli that have been transformed with the vector, 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.

One skilled m the art would also recognize that other vectors could be used. 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.

Once a suitable vector has been constructed, and transformed mto an appropriate Agrobacteri um strain, 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. This involves culturing plant ceils or tissues from the target plant. Generally, seeds from the plant targeted for transformation are collected, sterilized, rinsed m distilled water and then germinated on an agar surface for approximately ^"72 hours m the dar at approximately 25°C. The seeds are then moved to a lighted area under approximately 80

PPFD at

24-26°C. 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.

If the vectors are binary vectors in the appropriate strain of Agrobacteri um tumefaciens, the excised plant parts, which are called explants, are co-cultivated with the bacteria. After the explants are co-cultivated with the Agrobacteri um t umefaci ens harboring the bmary plasmid, they are transferred to a regeneration medium that is supplemented with an appropriate antibiotic, such as carbenicillm, to eliminate the bacteria. After a period of approximately two to four weeks, 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. Once the transgenic plant cells or tissues have been identified and separated from the non-transgenic cells or tissues and calli, they are regenerated mto plants.

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.

The inventors have also found that transgenic plants containing the phytoene synthase gene develop fruit color earlier and taste different than non-transgenic fruit.

By way of example, and not limitation, an example of the present invention will now be given.

EXAMPLE Construction of a binary vector

This example describes how to prepare a binary vector referred to as pETO203. 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. Those skilled in the art will recognize that alternative cloning strategies to those described herein can be used to clone these genetic elements mto a bmary vector. For a general molecular biology technique reference, refer to Sambrook et al. , (1989) Molecular Cloning: A Laboratory Manual. pETO203 has been deposited with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland, 20852. pETO203 was deposited with the ATCC on September 15, 1995, and assigned ATCC Number 97282.

Cloning of the tomato E8 promoter

The cloning of the E8 promoter from tomato { Lycopersicon escul entum) has been reported using standard molecular biology techniques (Lincoln and Fischer, (1987) PNAS 84:2793-2797) . The nucleotide sequence of various portions of the E8 promoter nas also been reported (Deικman and Fischer, (1988) EMBO J. 7:3315-3320; and Deikman et al., (1992) Plant Phvsiol. 100:2013-2017)

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'

(SEQUENCE ID. NO. 7) E8 primer #2 5' GCT TTC CAT GGT CTT TTG CA 3'

(SEQUENCE ID. NO. 8)

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. As a consequence of these mismatches, PCR amplification of this region of the tomato genomic DNA using E8 primers 1 and 2 resulted in the incorporation of an Ncol restriction site surrounding the initiation codon. Digestion of the amplification product with the restriction enzymes EcoRI and Ncol facilitate the directional cloning of the E8 promoter mto a binary vector with other genetic elements described below. 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. During import into the plastid, the ammo terminal extension, referred to as the targeting signal or plastid targeting signal (PTS , is removed Py enzymatic cleavage. In plant, 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.

To target the Erwinia herbicol a phytoene synthase gene product to the plastid, a PTS from the small subunit of the rιbulose-1, 5-bιsphosphate carboxylase oxygenase (RUBISCO) gene can be fused to the phytoene synthase gene. In a similar fashion to the cloning of the E8 promoter, 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. For the purpose of illustration, 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.

Cloning of the Erwinia herbicola phytoene synthase gene

The sequence of genes involved carotenoid biosynthesis from E. herbicola has been reported (Genbank Accession M87280) . In a similar manner to the cloning of the E8 promoter, the 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. For the purpose of illustration, 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 (NOS-T) 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. Cloning of genetic elements into a binary vector 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. In the cloning strategy described herein, 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.

To produce a derivative vector of pET0183 that would contam, m this order, the 1.1 kbp E8 promoter, the PTS, the crtB gene and nopalme synthase termination signals, a ligation reaction can be prepared and would include the following DNA fragments:

1. Parent Vector: 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

University, Piscataway, N.J.i digested with the restriction enzymes Xbal and EcoRI.

2. 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.

3. Targeting signal: The PCR-generated PTS DNA digested with Ncol and SphI.

4. Phytoene synthase gene: The PCR-generated DNA contammg the phytoene synthase gene digested with SphI and SacI .

5. NOS-T: pBI121 digested with SacI and EcoRI and the approximately 300 base pair fragment gel-purified.

After the ligation reaction, an aliquot can be taken and used to transform E. coli . Transformed colonies harboring the plasmid of interest can be identified by growing the culture and isolating the plasmid DNA. Several diagnostic restriction digests of these DNAs will show whether the various genetic elements have been fused together m the proper orientation. Once putative clones containing the above mentioned genetic elements (#2-5) have been identified by restriction digestion, one can precisely determine the integrity of the insertion by sequencing the inserted Xbal-EcoRI fragment. Plant transformation using vector pETO203

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 Phytacon™ 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 Agroύacteri 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. Next, they were incubated for 20 mmutes with bacterial inoculum containing 5 x IO⁸ CFU/mL of Agrobacteri um tumefaci ens, LBA440 : :pETO203, blotted dry, and cultured on the RIF co-cocultivation medium for 48 hours, at 24°C, in the dark.

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„-,_0St ,) on a shaker at 28°C, 180 rpm, for 24 hours.

Bacteria were then pelleted by centrifuging at 8000 rpm for 10 minutes in a Beckman J2-21 centrifuge using a JA-20 rotor. The bacterial pellet fraction was resuspended in a sterile MS medium to a concentration of 5 x 10⁸ CFU/mL using a spectrophotometric optical density reading at 550 nm (0.1 OD550=2xlO^e CFU/mL) . Prior to co-cultivation, the inoculum was supplemented with Acetsyringone (3' 5'dιmethoxy- 4 'hydroxy-acetophenone, Sigma, St. Louis, MO) to a final concentration of 600 micromolar.

Regeneration of transformed tomato plants

Five hundred tomato explants from an inbred line T7 were cocultivated with Agrobacteri um t umefaci ens LBA4404 : :pETO203 carrymg a plastid targeting signal and the phytoene synthase gene from Erwini a herbi col a driven by the 1.1 kbp E8 promoter. After two days of cocultivation, explants were transferred to a Rl/2 300 regeneration medium supplemented with 300 mg/L carbenicillm to eliminate the bacteria. After two weeks of culture explants were moved to fresh Rl/2 300 medium. Sectors of orange-pigmented callus became clearly visible on the callusing edges of the explants about one week after the last transfer. These pigmented sectors were cut out and subjected to a standard regeneration protocol. The remaining non-pigmented callus was also carried through a standard regeneration protocol. 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.

At the completion of the experiment a total of 377 tomato plants were regenerated. Among them, 85 plants were regenerated from the pigmented sectors and 292 plants were regenerated from the non-pigmented callus. All regenerated plants were indexed for presence of the phytoene synthase gene. Among the 85 plants regenerated from the orange callus, 42 plants were confirmed to be transgenic. This result indicates that expression of the phytoene synthase gene can aid identification and isolation of transgenic plants .

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.

Visual identification of proprietary transgenic plants.

Seeds from transgenic inbred line T7, transformed with the phytoene synthase gene from Erwinia herbicola driven by the E8 promoter (T7/E8PS), as described above and from nontransgenic control T7 inbred line were 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^Η 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.

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 photopeπod. 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.

The presence of orange pigmented callus allowed for rapid and unequivocal identification of explants from T7/E8PS plants transformed with the phytoene synthase gene. Media Used

All media used consisted of Murashigi and Skoog (Murashigi T., Skoog F., (1962) Phvsiol. Plant 15:437-498) salts and RO vitamins (composition listed below) , which were adjusted to pH=5.7 and solidified with 9 g/L of Noble Agar (Gibco) . Medium R1F was supplemented with 1 mg/L mdoleacetic acid (IAA), 0.65 mg/L zeat e and 16 g/L glucose. Medium Rl/2 was supplemented with 0.5 mg/L IAA, 0.325 mg/L of zeatme and 16 g/L glucose, medium RO was supplemented with 16 g/L glucose.

Table 1.

Composition of media used tomato regeneration. MS Salts mg/L

Ammonium nitrate 1650.000

Boric Acid 6.200 Calcium chloride 440.000 Cobaltous chloride 0.025

Cupric sulfate pentahydrate 0.025

Ferrous sulfate septahydrate 27.800

Magnesium sulfate septahydrate 370.000 Manganese sulfate monohydrate 15.600

Potassium iodide 0.083

Potassium nitrate 1900.000

Potassium phosphate monobasic 170.000

Sodium ethylenediamine tetraacetate 37.300 Sodium molybdate dihydrate 0.250

Zinc sulfate septahydrate 8.600

RO Vitamins mg/L

Nicotmic acid 5.000

Thiamme HCl 0.500 Pyridox e 0.500

Myo-mositol 100.000

Glycine 2.000

Although the invention has been described primarily m connection with the special and preferred embodiments, it will be understood that it is capable of modification without departing from the scope of the invention. The following claims are intended to cover all variations, uses or adaptions of the invention, following, in general, the principles thereof and mcludmg such departures from the presented disclosure as come with known or customary practice the field to which the invention pertains, or as are obvious to persons skilled in the field.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Trulson, Anna J. Braun III, Carl J. ii) TITLE OF INVENTION: A Method for Visually Selecting Transformed Plants Cells or Tissues by Carotenoid Pigmentation

(iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS :

(A) ADDRESSEE: Greer, Burns & Crain, Ltd. (B) STREET: 233 South Wacker Drive, Suite 8660, Sears Tower

(C) CITY: Chicago (D) STATE: IL

(E) COUNTRY: USA

(F) ZIP: 60606

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US (B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Mueller, Lisa V.

(B) REGISTRATION NUMBER: 38,978 (C) REFERENCE/DOCKETNUMBER: 1605.60833

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 312-993-0080

(B) TELEFAX: 312-993-0633

(2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2208 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE : DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

GAATTCATTT TTGACATCCC TAATGATATT GTTCACGTAA TTAAGTTTTG TGGAAGTGAG

60

AGAGTCCAAT TTTGATAAGA AAAGAGTCAG AAAACGTAAT ATTTTAAAAG TCTAAATCTT 120

TCTACAAATA AGAGCAAATT TATTTATTTT TTAATCCAAT AAATATTAAT GGAGGACAAA

180

TTCAATTCAC TTGGTTGTAA AATAAACTTA AACCAATAAC CAAAGANCTA ATAAATCTGA

240 AGTGGAATTA TTAAGGATAA TGTACATAGA CAATGAAGAA ATAATAGGTT CGATGAATTA

300

ATAATAATTA AGGATGTTAC AATCATCATG TGCCAAGTAT ATACACAATA TTCTATGGGA

360

TTTATAATTT CGTTACTTCA CTTAACTTTT GCGTAAATAA AACGAATTAT CTGATATTTT 420

ATAATAAAAC AGTTAATTAA GAACCATCAT TTTTAACAAC ATAGATATAT TATTTCTAAT

480

AGTTTAATGA TACTTTTAAA TCTTTTAAAT TTTATGTTTC TTTTAGAAAA TAAAAATTCA

540 AAAAAATTAA ATATATTTAC AAAAACTACA ATCAAACACA ACTTCATATA TTAAAAGCAA

600

AATATATTTT GAAAATTTCA AGTGTCCTAA CAAATAAGAC AAGAGGAAAA TGTACGATGA

660

GAGACATAAA GAGAACTAAT AATTGAGGAG TCCTATAATA TATAATAAAG TTTATTAGTA 720

AACTTAATTA TTAAGGACTC CTAAAATATA TGATAGGAGA AAATGAATGG TGAGAGATAT

780

TGGAAAACTT AATAATTAAG GATNTTAAAA TATATGGTAA AAGATAGGCA AAGTATCCAT

840 TATCCCCTTT TAACTTGAAG TCTACCTAGG CGCATGTGAA AGGTTGATTT TTTGTCACGT

900

CATATAGCTA TAACGTAAAA AAAGAAAGTA AAATTTTTAA TTTTTTTTAA TATATGACAT

960 ATTTTAAACG AAATATAGGA CAAAATGTAA ATGAATAGTA AAGGAAACAA AGATTAATAC

1020

TTACTTTGTA AGAATTTAAG ATAAATTTAA AATTTAATAG ATCAACTTTA CGTCTAGAAA

1080

GACCCATATC TAGAAGGAAT TTCACGAAAT CGGCCCTTAT TCAAAAATAA CTTTTAAATA 1140

ATGAATTTTA AATTTTAAGA AATAATATCC AATGAATAAA TGACATGTAG CATTTTACCT 1200

AAATATTTCA ACTATTTTAA TCCAATATTA ATTTGTTTTA TTCCCAACAA TAGAAAGTCT

1260

TGTGCAGACA TTTAATCTGA CTTTTCCAGT ACTAAATATT AATTTTCTGA AGATTTTCGG 1320

GTTTAGTCCA CAAGTTTTAG TGAGAAGTTT TGCTCAAAAT TTTAGGTGAG AAGGTTTGAT

1380

ATTTATCTTT TGTTAAATTA ATTTATCTAG GTGACTATTA TTTATTTAAG TAGAAATTCA

1440 TATCATTACT TTTGCCAACT TGTAGTCATA ATAGGAGTAG GTGTATATGA TGAAGGAATA

1500

AACAAGTTCA GTGAAGTGAT TAAAATAAAA TATAATTTAG GTGTACATCA AATAAAAACC

1560

TTAAAGTTTA GAAAGGCACC GAATAATTTT GCATAGAAGA TATTAGTAAA TTTATAAAAA 1620

TAAAAGAAAT GTAGTTGTCA AGTTGTCTTC TTTTTTTTGG ATAAAAATAG CAGTTGGCTT

1680

ATGTCATTCT TTTACAACCT CCATGCCACT TGTCCAATTG TTGACACTTA ACTAATTAGT

1740 TTGATTCATG TATGAATACT AAATAATTTT TTAGGACTGA CTCAAATATT TTTATATTAT

1800

CATAGTAATA TTTATCTAAT TTTTAGGACC ACTTATTACT AAATAATAAA TTAACTACTA

1860

CTATATTATT GTTGTGAAAC AACAACGTTT TGGTTGTTAT GATGAAACGT ACACTATATC 1920

AGTATGAAAA ATTCAAAACG ATTAGTATAA ATTATATTGA AAATTTGATA TTTTTCTATT

1980

CTTAATCAGA CGTATTGGGT TTCATATTTT AAAAAGGGAC TAAACTTAGA AGAGAAGTTT

2040 GTTTGAAACT ACTTTTGTCT CTTTCTTGTT CCCATTTCTC TCTTAGATTT CAAAAAGTGA

2100

ACTACTTTAT CTCTTTCTTT GTTCACATTT TATTTTATTC TATTATAAAT ATGGCATCCT

2160

CATATTGAGA TTTTTAGAAA TTATTCTAAT CATTCACAGT GCAAAAGA 2208

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 930 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS : unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..933

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :

ATG CGC CAA CCG CCG CTG CTT GAC CAC GCC ACG CAG ACC ATG GCC AAC

48 Met Arg Gin Pro Pro Leu Leu Asp His Ala Thr Gin Thr Met Ala Asn 1 5 10 15 GGC TCG AAA AGT TTT GCC ACC GCT GCG AAG CTG TTC GAC CCG GCC ACC

96 Gly Ser Lys Ser Phe Ala Thr Ala Ala Lys Leu Phe Asp Pro Ala Thr 20 25 30

CGC CGT AGC GTG CTG ATG CTC TAC ACC TGG TGC CGC CAC TGC GAT GAC 144

Arg Arg Ser Val Leu Met Leu Tyr Thr Trp Cys Arg His Cys Asp Asp 35 40 45

GTC ATT GAC GAC CAG ACC CAC GGC TTC GCC AGC GAG GCC GCG GCG GAG

192 Val lie Asp Asp Gin Thr His Gly Phe Ala Ser Glu Ala Ala Ala Glu 50 55 60

GAG GAG GCC ACC CAG CGC CTG GCC CGG CTG CGC ACG CTG ACC CTG GCG

240 Glu Glu Ala Thr Gin Arg Leu Ala Arg Leu Arg Thr Leu Thr Leu Ala 65 70 75 80

GCG TTT GAA GGG GCC GAG ATG CAG GAT CCG GCC TTC GCT GCC TTT CAG

288 Ala Phe Glu Gly Ala Glu Met Gin Asp Pro Ala Phe Ala Ala Phe Gin

85 90 95 GAG GTG GCG CTG ACC CAC GGT ATT ACG CCC CGC ATG GCG CTC GAT CAC

336 Glu Val Ala Leu Thr His Gly lie Thr Pro Arg Met Ala Leu Asp His 100 105 110

CTC GAC GGC TTT GCG ATG GAC GTG GCT CAG ACC CGC TAT GTC ACC TTT 384

Leu Asp Gly Phe Ala Met Asp Val Ala Gin Thr Arg Tyr Val Thr Phe 115 120 125

GAG GAT ACG CTG CGC TAC TGC TAT CAC GTG GCG GGC GTG GTG GGT CTG

432 Glu Asp Thr Leu Arg Tyr Cys Tyr His Val Ala Gly Val Val Gly Leu 130 135 140

ATG ATG GCC AGG GTG ATG GGC GTG CGG GAT GAG CGG GTG CTG GAT CGC

480 Met Met Ala Arg Val Met Gly Val Arg Asp Glu Arg Val Leu Asp Arg 145 150 155 160 GCC TGC GAT CTG GGG CTG GCC TTC CAG CTG ACG AAT ATC GCC CGG GAT

528 Ala Cys Asp Leu Gly Leu Ala Phe Gin Leu Thr Asn lie Ala Arg Asp 165 170 175 ATT ATT GAC GAT GCG GCT ATT GAC CGC TGC TAT CTG CCC GCC GAG TGG

576 lie lie Asp Asp Ala Ala lie Asp Arg Cys Tyr Leu Pro Ala Glu Trp 180 185 190

CTG CAG GAT GCC GGG CTG ACC CCG GAG AAC TAT GCC GCG CGG GAG AAT 624

Leu Gin Asp Ala Gly Leu Thr Pro Glu Asn Tyr Ala Ala Arg Glu Asn 195 200 205

CGC CCC GCG CTG GCG CGG GTG GCG GAG CGG CTT ATT GAT GCC GCA GAG

672 Arg Pro Ala Leu Ala Arg Val Ala Glu Arg Leu lie Asp Ala Ala Glu 210 215 220

CCG TAC TAC ATC TCC TCC CAG GCC GGG CTA CAC GAT CTG CCG CCG CGC

720 Pro Tyr Tyr lie Ser Ser Gin Ala Gly Leu His Asp Leu Pro Pro Arg 225 230 235 240

TGC GCC TGG GCG ATC GCC ACC GCC CGC AGC GTC TAC CGG GAG ATC GGT

768 Cys Ala Trp Ala lie Ala Thr Ala Arg Ser Val Tyr Arg Glu lie Gly 245 250 255 ATT AAG GTA AAA GCG GCG GGA GGC AGC GCC TGG GAT CGC CGC CAG CAC

816 lie Lys Val Lys Ala Ala Gly Gly Ser Ala Trp Asp Arg Arg Gin His 260 265 270

ACC AGC AAA GGT GAA AAA ATT GCC ATG CTG ATG GCG GCA CCG GGG CAG 864

Thr Ser Lys Gly Glu Lys lie Ala Met Leu Met Ala Ala Pro Gly Gin 275 280 285

GTT ATT CGG GCG AAG ACG ACG AGG GTG ACG CCG CGT CCG GCC GGT CTT

912 Val lie Arg Ala Lys Thr Thr Arg Val Thr Pro Arg Pro Ala Gly Leu 290 295 300

TGG CAG CGT CCC GTT TAG

930 Trp Gin Arg Pro Val * 305 310

(2) INFORMATION FOR SEQ ID NO: 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 311 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE : protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

Met Arg Gin Pro Pro Leu Leu Asp His Ala Thr Gin Thr Met Ala Asn 1 5 10 15

Gly Ser Lys Ser Phe Ala Thr Ala Ala Lys Leu Phe Asp Pro Ala Thr 20 25 30

Arg Arg Ser Val Leu Met Leu Tyr Thr Trp Cys Arg His Cys Asp Asp 35 40 45

Val lie Asp Asp Gin Thr His Gly Phe Ala Ser Glu Ala Ala Ala Glu 50 55 60 Glu Glu Ala Thr Gin Arg Leu Ala Arg Leu Arg Thr Leu Thr Leu Ala 65 70 75 80

Ala Phe Glu Gly Ala Glu Met Gin Asp Pro Ala Phe Ala Ala Phe Gin

85 90 95

Glu Val Ala Leu Thr His Gly lie Thr Pro Arg Met Ala Leu Asp His 100 105 110

Leu Asp Gly Phe Ala Met Asp Val Ala Gin Thr Arg Tyr Val Thr Phe 115 120 125

Glu Asp Thr Leu Arg Tyr Cys Tyr His Val Ala Gly Val Val Gly Leu 130 135 140 Met Met Ala Arg Val Met Gly Val Arg Asp Glu Arg Val Leu Asp Arg 145 150 155 160

Ala Cys Asp Leu Gly Leu Ala Phe Gin Leu Thr Asn lie Ala Arg Asp 165 170 175 lie lie Asp Asp Ala Ala lie Asp Arg Cys Tyr Leu Pro Ala Glu Trp 180 185 190

Leu Gin Asp Ala Gly Leu Thr Pro Glu Asn Tyr Ala Ala Arg Glu Asn 195 200 205

Arg Pro Ala Leu Ala Arg Val Ala Glu Arg Leu lie Asp Ala Ala Glu 210 215 220 Pro Tyr Tyr lie Ser Ser Gin Ala Gly Leu His Asp Leu Pro Pro Arg 225 230 235 240

Cys Ala Trp Ala lie Ala Thr Ala Arg Ser Val Tyr Arg Glu lie Gly 245 250 255 lie Lys Val Lys Ala Ala Gly Gly Ser Ala Trp Asp Arg Arg Gin His 260 265 270

Thr Ser Lys Gly Glu Lys lie Ala Met Leu Met Ala Ala Pro Gly Gin 275 280 285

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

(2) INFORMATION FOR SEQ ID NO:4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 171 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..171

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 :

ATG GCT TCC TCA GTT CTT TCC TCT GCA GCA GTT GCC ACC CGC AGC AAT

48 Met Ala Ser Ser Val Leu Ser Ser Ala Ala Val Ala Thr Arg Ser Asn 1 5 10 15

GTT GCT CAA GCT AAC ATG GTG GCG CCT TTC ACT GGC CTT AAG TCA GCT

96 Val Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Ala 20 25 30

GCC TCA TTC CCT GTT TCA AGG AAG CAA AAC CTT GAC ATC ACT TCC ATT

144 Ala Ser Phe Pro Val Ser Arg Lys Gin Asn Leu Asp lie Thr Ser lie 35 40 45 GCC AGC AAC GGC GGA AGA GTG CAA TGC

171 Ala Ser Asn Gly Gly Arg Val Gin Cys 50 55

(2) INFORMATION FOR SEQ ID NO: 5 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 57 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 :

Met Ala Ser Ser Val Leu Ser Ser Ala Ala Val Ala Thr Arg Ser Asn 1 5 10 15

Val Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Ala 20 25 30 Ala Ser Phe Pro Val Ser Arg Lys Gin Asn Leu Asp lie Thr Ser lie 35 40 45 Ala Ser Asn Gly Gly Arg Val Gin Cys 50 55

(2) INFORMATION FOR SEQ ID NO: 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 263 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE : DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 :

GAATTTCCCC GATCGTTCAA ACATTTGGCA ATAAAGTTTC TTAAGATTGA ATCCTGTTGC

60

CGGTCTTGCG ATGATTATCA TATAATTTCT GTTGAATTAC GTTAAGCATG TAATAATTAA

120 CATGTAATGC ATGACGTTAT TTATGAGATG GGTTTTTATG ATTAGAGTCC CGCAATTATA

180

CATTTAATAC GCGATAGAAA ACAAAATATA GCGCGCAAAC TAGGATAAAT TATCGCGCGC

240

GGTGTCATCT ATGTTACTAG ATC 263

(2) INFORMATION FOR SEQ ID NO:7 :

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS : unknown

(D) TOPOLOGY: unknown ui) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7

NNGAATTCAT TTTTGACATC 20

(2) INFORMATION FOR SEQ ID NO: 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS : unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 :

GCTTTCCATG GTCTTTTGCA

20

Claims

1. A method for visually identifying transgenic plant cells or tissues from non-transgenic plant cells or tissues by carotenoid pigmentation, the method comprising the steps of: a) culturing transgenic plant and non-transgenic plant cells or tissues in a culture medium, wherem said transgenic cells or tissues contam a heterologous, recombinant chimeric DNA segment containing at least one expression cassette provided that at least one expression cassette comprises:

(1) a promoter DNA segment which functions in plant cells or tissues;

(2) a DNA segment comprising a plastid targeting signal fused to the coding region of the phytoene synthase gene of the Erwinia genus group of genes which express a colored member of the biosynthetic pathway, wherein said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results in the production of carotenoid pigmentation in plant cells or tissues; and

(3) a 3' non-translated DNA segment which termmates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; b) growing said transgenic and non-transgenic cells or tissues for a sufficient amount of time in said medium to allow the transgenic cells or tissues to express the phytoene synthase gene; and c) identifying the transgenic cells or tissues by their carotenoid pigmentation. 2. A method for visually identifying transgenic plant cells or tissues from non-transgenic plant cells or tissues by carotenoid pigmentation, the method comprising tne steps of: a_; culturing transgenic and non-transgenic plant cells or tissues in a culture medium, wherein said transgenic plant cells or tissues contain a heterologous, recombinant chimeric DNA segment containing at least two expression cassettes, wherein the first expression cassette comprises:

(1) a promoter DNA segment which functions plant cells or tissues;

(2) a DNA segment comprising a plastid targeting signal fused to the coding region of the phytoene synthase gene of the Erwi nia genus group of genes which express a colored member of the biosynthetic pathway, wherein said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results in the production of carotenoid pigmentation in plant cells or tissues; and (3) a 3' non-translated DNA segment which terminates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; and the second expression cassette comprises:

(1) a promoter DNA segment which functions plant cells or tissues;

(2) a DNA segment which encodes a second gene which produces RNA which encodes a target protein; and

(3) a 3' non-translated DNA segment which termmates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; b) growing said transgenic and non-transgenic cells or tissues for a sufficient amount of time in said medium to allow the transformed cells or tissues to express the phytoene synthase gene; and c) identifying the transformed cells or tissues by their carotenoid pigmentation.

3. The method of claims 1 or 2 wherein the Erwi ni a genus is selected from the group consisting of: Erwi ni a uredovora and Erwinia herbi cola .

4. A method for visually identifying transgenic plant cells or tissues from non-transgenic plant cells or tissues by carotenoid pigmentation, the method comprising the steps of: a) culturing transgenic and non-transgenic plant cells or tissues in a culture medium, wherein said transgenic plant cells or tissues contam a heterologous, recombinant chimeric DNA segment containing at least one expression cassette provided that at least one expression cassette comprises:

(1) a promoter DNA segment which functions m plant cells or tissues;

(2) a DNA segment comprising a plastid targeting signal fused to the coding region of the phytoene synthase gene of the Erwinia genus group from Erwinia herbicola, wherein said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results m the production of carotenoid pigmentation in plant cells or tissues; and

(3) a 3' non-translated DNA segment which termmates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; b) growing said transgenic and non-transgenic cells or tissues for a sufficient amount of time in said medium to allow the transformed cells to express the phytoene synthase gene; and ci identifying the transgenic cells or tissues by their carotenoid pigmentation. Ξ. A method for visually identifying transgenic plant cells or tissues from non-transgenic plant cells or tissues by carotenoid pigmentation, the method comprising tne steps of: a culturing transgenic and transgenic plant cells or tissues m a culture medium, wherein said transgenic plant cells or tissues contain a heterologous, recombinant chimeric DNA segment contammg at least two expression cassettes, wherein the first expression cassette comprises:

(1) a promoter DNA segment which functions in plant cells or tissues;

(2) a DNA segment comprismg a plastid targeting signal fused to the coding region of the phytoene synthase gene from Erwinia herbicola , wherein said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results in the production of carotenoid pigmentation plant cells or tissues; and

(3) a 3' non-translated DNA segment which terminates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; and the second expression cassette comprises:

(1) a promoter DNA segment which functions in plant cells or tissues; (2) a DNA segment which encodes a second gene which produces RNA which encodes a target protein; and

(3) a 3¹ non-translated DNA segment which terminates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; b) growing said transgenic and non-transgenic cells or tissues for a sufficient amount of time m said medium to allow the transgenic cells or tissues to express the phytoene synthase gene; and c) identifying the transformed cells or tissues by their carotenoid pigmentation.

6. The method of claims 1, 2, 4 or 5 wherein the recombinant DNA sequence is introduced into the plant cells or tissues by microprojectile bombardment.

7. The method of claims 1, 2, 4 or 5 wherein the recombinant DNA sequence is introduced mto the plant cells or tissues by microinjection.

8. The method of claims 1, 2, 4 or 5 wherein the recombinant DNA sequence is introduced mto a vector. 9. The method of claim 8 wherein the vector is a bmary vector in an appropriate strain of Agrobacterium tumefaci ens .

10. The method of claim 8 wherein the vector is a co tegratmg vector. 11. The method of claim 9 wherein the binary vector from Agrobacteri um tumefaciens is introduced mto the plant cells via Agrobacteriurn-mediated gene transfer.

12. The method of claims 1, 2, 4 or 5 wherein the plant cells or tissues are selected from the group consisting of tomato, cucurbits, pepper, lettuce and carrots.

13. A method for selectively growing transgenic plants from a mixture of transgenic and non-transgenic plant cells or tissues comprising the steps of: a) culturing transgenic and non-transgenic plant cells or tissues m a culture medium, wherein said transgenic plant cells or tissues contam a heterologous, recombinant chimeric DNA segment contammg at least one expression cassette provided that at least one expression cassette comprises:

(1) a promoter DNA segment which functions in plant cells or tissues;

(2) a DNA segment comprismg a plastid targeting signal fused to the coding region of the phytoene synthase gene of the Erwini a genus group of genes which express a colored member of the biosynthetic pathway, wherein said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results m the production of carotenoid pigmentation in plant cells or tissues; and (3) a 3' non-translated DNA segment which terminates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; b) growing said transgenic and non-transgenic cells or tissues for a sufficient amount of time in said medium to allow the transgenic cells to or tissues express the phytoene synthase gene; and c) identifying the transgenic cells or tissues by their carotenoid pigmentation; d) recovering the transgenic cells or tissues; and e) regenerating plants from said transgenic cells or tissues .

14. A method for selectively growing transgenic plants from a mixture of transgenic and non-transgenic plant cells or tissues comprising the steps of: a) culturing transgenic and non-transgenic plant cells or tissues m a culture medium, wherein said transgenic plant cells or tissues contam a heterologous, recombinant chimeric DNA segment contammg at least two expression cassettes, wherein the first expression cassette comprises:

(1) a promoter DNA segment which functions in plant cells or tissues;

(2) a DNA segment comprising a plastid targeting 5 signal fused to the coding region of the phytoene synthase gene of the Erwini a genus group of genes which express a colored member of the biosynthetic pathway, wherein said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results in the production C of carotenoid pigmentation in plant cells or tissues; and

(3) a 3' non-translated DNA segment which terminates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; and tne second expression cassette comprises: (1) a promoter DNA segment which functions in plant cells or tissues;

(2) a DNA segment which encodes a second gene which produces RNA which encodes a target protein; and (3) a 3' non-translated DNA segment which termmates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; b) growing said said transgenic and non-transgenic cells or tissues for a sufficient amount of time said medium to allow the transgenic cells or tissues to express the phytoene synthase gene; and c) identifying the transgenic cells or tissues by their carotenoid pigmentation; d) recovering the transgenic cells or tissues; and e) regenerating plants from said transgenic cells or tissues .

15. The method of claims 13 or 14 wherein the Erwi ni a genus is selected from the group consisting of: Erwinia uredovora and Erwinia herbi cola . 16. A method for selectively growing transgenic plants from a mixture of transgenic and non-transgenic plant cells or tissues comprising the steps of: a) culturing transgenic and non-transgenic plant cells or tissues m a culture medium, wherein said transgenic cells or tissues contam a heterologous, recombinant chimeric DNA segment containing at least one expression cassette provided that at least one expression cassette comprises:

(1) a promoter DNA segment which functions m pj_ant cells or tissues;

(2) a DNA segment comprising a plastid targeting signal fused to tne coding region of the phytoene synthase gene of the Erwinia genus group from Erwinia hervi cola , where said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results in the production of carotenoid pigmentation in plant cells or tissues; and

(3) a 3' non-translated DNA segment which terminates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; b) growing said said transgenic and non-transgenic cells or tissues for a sufficient amount of time in said medium to allow the transgenic cells or tissues to express the phytoene synthase gene; and c) identifying the transgenic cells or tissues by their carotenoid pigmentation; d) recovering the transgenic cells or tissues; and e) regenerating plants from said transgenic cells or tissues.

17. A method for selectively growing transgenic plants from a mixture of transgenic and non-transgenic plant cells or tissues comprising the steps of: a) culturing transgenic and non-transgenic plant cells or tissues in a culture medium, wherein said transgenic cells or tissues contam a heterologous, recombinant chimeric DNA segment containing at least two expression cassettes, wherein the first expression cassette comprises: (1) a promoter DNA segment which functions plant cells or tissues;

(2) a DNA segment comprising a plastid targeting signal fused to the coding region of the phytoene synthase gene from Erwi ni a herbi col a , wherein said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results m the production of carotenoid pigmentation m plant cells or tissues; and

(3) a 3' non-translated DNA segment which termmates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; and the second expression cassette comprises:

(3) a 3' non-translated DNA segment which termmates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; b) growing said said transgenic and non-transgenic cells or tissues for a sufficient amount of time m said medium to allow the transgenic cells or tissues to express the phytoene synthase gene; and c) identifying the transgenic cells or tissues by their carotenoid pigmentation; d) recovering the transgenic cells or tissues; and e) regenerating plants from said transgenic cells or tissues .

18. The method of claims 13, 14, 16 or 17, wherein the plant is selected from the group consisting of: tomato, cucurbits, pepper, lettuce and carrots.

19. A method for identifying proprietary transgenic plants, the method comprising the steps of: a) supplying at least one proprietary transgenic plant to be identified; bi culturing explants from said proprietary plant m a culture medium, wherein said proprietary transgenic plant contains a heterologous, recombinant chimeric DNA segment containing at least one expression cassette provided that at least one expression cassette comprises:

(1) a promoter DNA segment which functions in plant cells or tissues;

(2) a DNA segment comprising a plastid targeting s-.gnaι fused to the coding region of the pnytoene synthase gene of the Erwinia genus group of genes which express a colored member of the biosynthetic pathway, wherein said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results in the production of carotenoid pigmentation in plant cells or tissues; and (3) a 3' non-translated DNA segment which termmates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; b) growing said said explants for a sufficient amount of time said medium to allow the transgenic cells or tissues to express the phytoene synthase gene; and c) identifying the proprietary transgenic cells or tissues by their carotenoid pigmentation.

20. A method for identifying proprietary transgenic plants, the method comprismg the steps of: a) supplying at least one proprietary transgenic plant to be identified; b) culturing explants from said proprietary transgenic plant in a culture medium, wherein said proprietary transgenic plant contains a heterologous, recombinant chimeric DNA segment containing at least two expression cassettes, wherein the first expression cassette comprises:

(1) a promoter DNA segment which functions m plant cells or tissues;

(2) a DNA segment comprising a plastid targeting signal fused to the coding region of the phytoene synthase gene of the Erwi ni a genus group of genes which express a colored member of the biosynthetic pathway, wherein said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results in the production of carotenoid pigmentation plant cells or tissues; and

(3) a 3' non-translated DNA segment which termmates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; b growing said said explants for a sufficient amount of time said medium to allow the transgenic cells or tissues to express the phytoene synthase gene; and c) identifying the proprietary transgenic cells by their carotenoid pigmentation. 21. The method of claims 19 or 20 wherein the

Erwi nia genus is selected from the group consisting of: Erwinia uredovora and Erwinia herbi cola .

22. A method for identifying proprietary transgenic plants, the method comprismg the steps of: a) supplying at least one proprietary transgenic plant to be identified; b) culturing explants from said proprietary plant a culture medium, wherein said proprietary transgenic plant contains a heterologous, recombinant chimeric DNA segment containing at least one expression cassette provided that at least one expression cassette comprises:

(1) a promoter DNA segment which functions m plant cells or tissues;

(2) a DNA segment comprising a plastid targeting signal fused to the coding region of the phytoene synthase gene of the Erwinia genus group from Erwinia herbicola, wherein said DNA segment produces RNA which encodes a chimeric polypeptide and said poiypeptide results the production of carotenoid pigmentation in plant cells and tissues; and

(3) a 3' non-translated DNA segment which termmates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; b) growing said said transgenic and non-transgenic cells or tissues for a sufficient amount of time in said medium to allow the transgenic cells to express the phytoene synthase gene; and c) identifying the proprietary transgenic cells oir tissues by their carotenoid pigmentation.

23. A method for identifying proprietary transgenic plants, the method comprising the steps of: a) supplying at least one proprietary transgenic plant to be identified; b) culturing explants from said proprietary plant in a culture medium, wherein said proprietary transgenic plant contains a heterologous, recombinant chimeric DNA segment contammg at least two expression cassettes, wherein the first expression cassette comprises: (1) a promoter DNA segment which functions in plant cells or tissues;

(2) a DNA segment comprising a plastid targeting signal fused to the coding region of the phytoene synthase gene from Erwini a herbi cola, wherein said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results in the production of carotenoid pigmentation m plant cells or tissues; and

(3) a 3' non-translated DNA segment which terminates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; and tne second expression cassette comprises:

(1) a promoter DNA segment which functions m plant cells or tissues;

(3) a 3' non-translated DNA segment which terminates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; 5 b) growing said said transgenic and non-transgenic cells or tissues for a sufficient amount of time m said medium to allow the transgenic cells to express the phytoene synthase gene; and c) identifying the proprietary transgenic cells or o tissues by their carotenoid pigmentation.

24. The method of claims 19, 20, 22 and 23, wherein the plants are selected from the group consisting of: tomato, cucurbits, pepper, lettuce and carrots.

25. A transgenic plant whose genome comprises a 5 heterologous chimeric DNA segment that contains at least one expression cassette provided that at least one expression cassette comprises:

(1) a promoter DNA segment which functions plant cells or tissues; 0 (2) a DNA segment comprising a plastid targeting signal fused to the coding region of the phytoene synthase gene of the Erwini a genus group of genes which express a colored member of the biosynthetic pathway, wherein said DNA 5 segment produces RNA which encodes a chimeric polypeptide and said polypeptide results m the production of carotenoid pigmentation plant cells or tissues; and

(3^ a 3' non-translated DNA segment which terminates C transcription ana adds a polyadenylated tract of residues to the 3' eno of the RNA segment.

26. A transgenic plant whose genome comprises a heterologous DNA segment that contains at least two expression cassettes wherein the first expression cassette comprises :

(1) a promoter DNA segment which functions in plant cells or tissues; (2) a DNA segment comprising a plastid targeting signal fused to the coding region of the phytoene synthase gene of the Erwinia genus group of genes which express a colored member of the biosynthetic pathway, wherein said

DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results the production of carotenoid pigmentation in plant cells or tissues; and (3) a 3' non-translated DNA segment which terminates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment; and the second expression cassette comprises:

(1) a promoter DNA segment which functions in plant cells or tissues;

(2) a DNA segment which encodes a second gene which produces RNA which encodes a target protein; and (3) a 3' non-translated DNA segment which terminates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment.

27. The method of claims 25 or 26 wherein the Erwinia genus is selected from the group consisting of: Erwini a uredovora and Erwinia herbi cola .

28. A transgenic plant whose genome comprises a heterologous chimeric DNA segment that contains at least one expression cassette provided that at least one expression cassette comprises: (1) a promoter DNA segment which functions m plant cells or tissues;

(2) a DNA segment comprising a plastid targeting signal fused to tne coding region of the phytoene synthase gene of the Erwini a genus group of Erwinia herbi cola where said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results in the production of carotenoid pigmentation in plant cells or tissues; and (3) a 3' non-translated DNA segment which terminates transcription and adds a polyadenylated tract of residues to the 3' end of the RNA segment.

29. A transgenic plant whose genome comprises a heterologous chimeric DNA segment that contains at least two expression cassettes wherein the first expression cassette comprises:

(1) a promoter DNA segment which functions plant cells or tissues;

(2) a DNA segment comprismg a plastid targeting signal fused to the coding region of the phytoene synthase gene of the Erwinia genus group of Erwinia herbicola, wherein said DNA segment produces RNA which encodes a chimeric polypeptide and said polypeptide results m the production of carotenoid pigmentation m plant cells; and

(3) a 3' non-translated DNA segment which termmates transcription and adds a polyadenylated tract of residues to the 3; end of the RNA segment; and the second expression cassette comprises: (1) a promoter DNA segment which functions m plant cells;

(2) a DNA segment which encodes a second gene which produces RNA which encodes a target protein; and '3) a 3' non-translated DNA segment which terminates transcription and adds a polyadenylated tract of residues to the 3' end of tne RNA segment.

30. The plants of claims 25, 26, 28 or 29 wherein the plant is selected from tne group consisting of: tomato, cucurbits, pepper, lettuce and carrots.

31. Seed from a transgenic plant according to claims 25, 26, 28 or 29.

32. A plasmid designated pETO203 having American Type Culture Collection accession number 97282.