Cotton modification using ovary-tissue transcriptional factors

Novel methods are provided whereby encoding sequences preferentially directing gene expression in ovary tissue, particularly in very early fruit development, are utilized to express genes encoding isopentenyl transferase in cotton ovule tissue. The methods permit the modification of the characteristics of boll set in cotton plants and provide a mechanism for altering fiber quality characteristics including fiber dimension and strength.

TECHNICAL FIELD
 This invention relates to methods of using in vitro constructed DNA
 transcription or expression cassettes capable of directing ovary-tissue
 transcription of a DNA sequence of interest in cotton plants to produce
 ovary-derived cells having an altered phenotype. The invention is
 exemplified by methods of using ovary tissue promoters for altering the
 phenotype of boll production in cotton plants and also for modifying the
 quality of cotton fibers. Included are cotton plants and cotton fibers
 produced by the method.
 BACKGROUND OF THE INVENTION
 1. Background
 The ability to manipulate characteristics of fiber quality in cotton
 through genetic engineering techniques would permit the rapid introduction
 of improved cotton varietes. Cotton fiber quality is conventionally
 measured in terms of characteristics of strength, length and micronaire (a
 measurement of fiber fineness).
 In general, genetic engineering techniques have been directed to modifying
 the phenotype of individual prokaryotic and eukaryotic cells, especially
 in culture. Plant cells have proven more intransigent than other
 eukaryotic cells, due not only to a lack of suitable vector systems but
 also as a result of the different goals involved. For many applications,
 it is desirable to be able to control gene expression at a particular
 stage in the growth of a plant or in a particular plant part. For this
 purpose, regulatory sequences are required which afford the desired
 initiation of transcription in the appropriate cell types and/or at the
 appropriate time in the plant's development without having serious
 detrimental effects on plant development and productivity. It is therefore
 of interest to be able to isolate sequences which can be used to provide
 the desired regulation of transcription in a plant cell during the growing
 cycle of the host plant.
 One aspect of this interest is the ability to change the phenotype of
 particular cell types, such as differentiated epidermal cells that
 originated in ovary tissue, so as to provide for altered or improved
 aspects of the mature cell type. In order to effect the desired phenotypic
 changes, transcription initiation regions capable of initiating
 transcription in early ovary development are used. These transcription
 initiation regions are active prior to the onset of pollination and are
 less active or inactive, before fruit enlargement, tissue maturation, or
 the like occur.
 2. Relevant Literature
 Methods and compositions for modulating cytokinin expression in tomato
 fruit are described in U.S. Pat. No. 5,177,307. U.S. Pat. No. 5,175,095
 describes ovary tissue transcriptional promoters, including a pZ7 promoter
 active in ovule integument cells. The disclosure of both patents is hereby
 incorporated by reference. Neither patent describes a method for modifying
 a characteristic of cotton fiber quality.
 A class of fruit-specific promoters expressed at or during anthesis through
 fruit development, at least until the beginning of ripening, is discussed
 in European Application 88.906296.4, the disclosure of which is hereby
 incorporated by reference. cDNA clones that are preferentially expressed
 in cotton fiber have been isolated. One of the clones isolated corresponds
 to mRNA and protein that are highest during the late primary cell wall and
 early secondary cell wall synthesis stages. John Crow Pro. Natl. Acad.
 Sci. (1992) 89:5769-5773. cDNA clones from tomato displaying differential
 expression during fruit development have been isolated and characterized
 (Mansson et al., Mol. Gen. Genet. (1985) 200:356-361: Slater et al., Plant
 Mol. Biol. (1985) 5: 137-147). These studies have focused primarily on
 mRNAs which accumulate during fruit ripening. One of the proteins encoded
 by the ripening-specific cDNAs has been identified as polygalacturonase
 (Slater et al., Plant Mol. Biol. (1985) 5:137-147). A cDNA clone which
 encodes tomato polygalacturonase has been sequenced (Grierson et al.,
 Nucleic Acids Research (1986) 14:8395-8603). Improvements in aspects of
 tomato fruit storage and handling through transcriptional manipulation of
 expression of the polygalacturonase gene have been reported (Sheehy et
 al., Proc. Natl. Acad. Sci. (1988) 85:8805-8809; Smith et al., Nature
 (1988) 334: 724-726).
 Mature plastid mRNA for psbA (one of the components of photosystem II)
 reaches its highest level late in fruit development, whereas after the
 onset of ripening, plastid mRNAs for other components of photosystem I and
 II decline to nondetectable levels in chromoplasts (Piechulla et al.,
 Plant Mol. Biol. (1986) 7:367-376). Recently, cDNA clones representing
 genes apparently involved in tomato pollen (McCormick et al., Tomato
 Biotechnology (1987) Alan R. Liss, Inc., NY) and pistil (Gasser et al.,
 Plant Cell (1989), 1:15-24) interactions have also been isolated and
 characterized.
 Other studies have focused on genes inducibly regulated, e.g. genes
 encoding serine proteinase inhibitors, which are expressed in response to
 wounding in tomato (Graham et al., J. Biol. Chem. (1985) 260:6555-6560:
 Graham et al., J. Biol. Chem. (1985) 260:6561-6554) and on mRNAs
 correlated with ethylene synthesis in ripening fruit and leaves after
 wounding (Smith et al., Planta (1986) 168: 94-100). Accumulation of a
 metallocarboxypeptidase inhibitor protein has been reported in leaves of
 wounded potato plants (Graham et al., Biochem & Biophys. Res. Comm. (1981)
 101: 1164-1170; Martineau et al., Mol. Gen. Genet. (1991) 228:281-286).
 Genes which are expressed preferentially in plant seed tissues, such as in
 embryos or seed coats, have also been reported. (See, for example,
 European Patent Application 87306739.1 (published as 0 255 378 on Feb. 3,
 1988) and Kridl et al., Seed Science Research (1991) 1:209-219).
 Agrobacterium-mediated cotton transformation is described in Umbeck, U.S.
 Pat. Nos. 5,004,863 and 5,159,135 and cotton transformation by particle
 bombardment is reported in WO 92/15675, published Sep. 17, 1992.
 Transformation of Brassica has been described by Radke et al., (Theor.
 Appl. Genet. (1988) 75:685-694; Plant Cell Reports (1992) 11:499-505).
 Transformation of cultivated tomato is described by McCormick et al., Plant
 Cell Reports (1986) 5:81-89 and Fillatti et al., Bio/Technology (1987)
 5:726-730.
 SUMMARY OF THE INVENTION
 The invention generally comprises the use of DNA constructs having a
 transcriptional and translational initiation region functional in ovule
 integument cells to express a DNA sequence encoding a protein active in
 the production of a plant growth substance in methods to alter the
 phenotype of cotton plants and/or cotton fiber cells. The term plant
 growth substance generally refers to compounds that elicit growth,
 developmental or metabolic responses in the plant. Such substances are not
 metabolites in the sense that they are not intermediates or products of
 the pathways they control, and they are active at very low concentrations.
 Some are active in promoting growth or development, while others function
 more as inhibitors of the same. As such, plant growth substances would
 include such substances as auxins, giberrellins, cytokinins, ethylene and
 abscissic acid, which are also often referred to as plant hormones.
 Proteins active in the production of a plant growth substance could include
 enzyme involved in the ethylene biosynthesis pathway. A number of such
 enzymes have been described, including ACC synthase, the ethylene forming
 enzyme (also referred to as pTOM13), SAM synthase, ACC deaminase and SAM
 decarboxylase.
 The method generally comprises growing a transgenic cotton plant to produce
 mature ovule tissue, wherein cells of the mature ovule tissue comprise in
 their genome such a construct. The construct also will have a
 transcriptional termination region as an additional component. At least
 one of the components will be exogenous to at least one other of said
 components, i.e., the construct components do not naturally occur together
 as a group. Under these circumstances the plant expresses the protein
 active in the production of a plant growth substance in mature plant ovule
 tissue.
 It has been discovered that the expression of a protein active in the
 production of a plant growth substance from such a construct can be used
 to alter the rate of boll production in a transgenic cotton plant.
 Exemplified is the expression of cytokinin in ovule integument cells to
 increase boll production.
 It has also been discovered that the fiber quality of a transgenic cotton
 plant may be modified by such a method. Particularly, the modification of
 characteristics of cotton fiber dimension, such as the length, strength or
 micronaire of the fiber are exemplified in the expression of cytokinin
 from the pZ7 transcriptional and translational initiation region.

DETAILED DESCRIPTION OF THE INVENTION
 In accordance with the subject invention, a method is provided for
 influencing the quality of fiber derived from a transgenic cotton plant.
 Also provided is a method whereby the modification of the rate of boll
 production in a transgenic cotton plant can be achieved. Constructs for
 use in the methods may include several forms, depending upon the intended
 use of the construct. Thus, the constructs include vectors,
 transcriptional cassettes, expression cassettes and plasmids. The
 transcriptional and translational initiation region (also sometimes
 referred to as a "promoter,"), preferably comprises a transcriptional
 initiation regulatory region and a translational initiation regulatory
 region of untranslated 5' sequences, "ribosome binding sites," responsible
 for binding mRNA to ribosomes and translational initiation. It is
 preferred that all of the transcriptional and translational functional
 elements of the initiation control region are derived from or obtainable
 from the same gene. In some embodiments, the promoter will be modified by
 the addition of sequences, such as enhancers, or deletions of nonessential
 and/or undesired sequences. By "obtainable" is intended a promoter having
 a DNA sequence sufficiently similar to that of a native promoter to
 provide for the desired specificity of transcription of a DNA sequence of
 interest. It includes natural and synthetic sequences as well as sequences
 which may be a combination of synthetic and natural sequences.
 The vectors typically comprise a nucleotide sequence of one or more
 nucleotides and a transcriptional initiation regulatory region associated
 with gene expression in ovary tissue. A transcriptional cassette for
 transcription of a nucleotide sequence of interest in ovary tissue will
 include in the direction of transcription, an ovary tissue transcriptional
 initiation region and optionally a translational initiation region, a DNA
 sequence of interest, and a transcriptional and optionally translational
 termination region functional in a plant cell. When the cassette provides
 for the transcription and translation of a DNA sequence of interest it is
 considered an expression cassette. One or more introns may also be
 present.
 Other sequences may also be present, including those encoding transit
 peptides and secretory leader sequences as desired. The regulatory regions
 are capable of directing transcription in ovary cells from anthesis
 through flowering but direct little or no expression after the initial
 changes which occur at the time surrounding pollination and/or
 fertilization; transcription from these regulatory regions is not
 detectable at about three weeks after anthesis. Further, ovary-tissue
 transcription initiation regions of this invention are typically not
 readily detectable in other plant tissues. Transcription initiation
 regions from ovary tissue that are not ovary specific may find special
 application. Especially preferred are transcription initiation regions
 which are not found at stages of fruit development other than perianthesis
 through flowering. Transcription initiation regions capable of initiating
 transcription in other plant tissues and/or at other stages of ovary
 development, in addition to the foregoing, are acceptable insofar as such
 regions provide a significant expression level in ovary tissue at the
 defined periods of interest and do not negatively interfere with the plant
 as a whole, and, in particular, do not interfere with the development of
 fruit and/or fruit-related parts. Also of interest are ovary tissue
 promoters and/or promoter elements which are capable of directing
 transcription in specific ovary tissues such as outer pericarp tissue,
 inner core tissues, integuments, and the like.
 Transcriptional initiation regions which are expressible in ovary tissue at
 or near maximal levels during the period of interest of this invention,
 generally the flowering period of plant reproductive cycles, are
 preferred. Of particular interest is the period of at least one to three
 days prior to anthesis through flower senescence. The transcription level
 should be sufficient to provide an amount of RNA capable of resulting in a
 modified fruit. The term "fruit" as used herein refers to the mature organ
 formed as the result of the development of the ovary wall of a flower and
 any other closely associated parts. See Weirer, T. E., 1, ed., Botany An
 Introduction to Plant Biology (6th ed.) (John Wiley & Sons, 1982); Tootill
 & Backmore, The Facts on File Dictionary of Botany (Market Home Books
 Ltd., 1984). By "modified fruit" is meant fruit having a detectably
 different phenotype from a nontransformed plant of the same species, for
 example, one not having the transcriptional cassette in question in its
 genome.
 Of particular interest are transcriptional initiation regions associated
 with genes expressed in ovary tissue and which are capable of directing
 transcription at least 24 hours prior to anthesis through flower
 senescence. The term "anthesis" refers herein to the period associated
 with flower opening and flowering. The term "flower senescence" refers
 herein to the period associated with flower death, including the loss of
 the (flower) petals, etc. Abercrombie, M., et al., A Dictionary of Biology
 (6th ed) (Penguin Books, 1973). Unopened flowers, or buds, are considered
 "pre-anthesis." Anthesis begins with the opening of the flower petals,
 which represents a sexually receptive portion of the reproductive cycle of
 the plant. Typically, flowering lasts approximately one week in the tested
 UCB82 tomato variety. In a plant like cotton, flowering lasts
 approximately two weeks and the fiber develops from the seed coat tissue.
 It is preferred that the transcriptional initiation regions of this
 invention do not initiate transcription for a significant time or to a
 significant degree prior to plant flower budding. Ideally, the level of
 transcription will be high for at least approximately one to three days
 and encompass the onset of anthesis ("perianthesis").
 It further is desired that the transcriptional initiation regions of this
 invention show a decreased level of transcriptional activity within 1-3
 days after the onset of anthesis which does not increase, and preferably
 decreases over time. Fertilization of a tomato embryo sac, to produce the
 zygote that forms the embryo plant, typically occurs 2-3 days after flower
 opening. This coincides with a decrease in the activity of a
 transcriptional initiation region of this invention. Thus, it is desired
 that the transcriptional activity of the promoter of this invention
 significantly decrease within about two days after the onset of anthesis.
 Transcriptional initiation regions of this invention will be capable of
 directing expression in ovary tissue at significant expression levels
 during the preferred periods described above.
 In some embodiments, it will be desired to selectively regulate
 transcription in a particular ovary tissue or tissues. When used in
 conjunction with a 5' untranslated sequence capable of initiating
 translation, expression in defined ovary tissue, including ovary
 integuments (also known as "ovule epidermal cells"), core or pericarp
 tissue, and the like, the transcriptional initiation region can direct a
 desired message encoded by a DNA sequence of interest in a particular
 tissue to more efficiently effect a desired phenotypic modification.
 Of special interest are transcription initiation regions expressible in at
 least ovary outer pericarp tissue. In cotton the analogous ovary structure
 is the burr of the cotton boll. Regulating expression in ovary integuments
 and/or core tissue has resulted in useful modifications to the boll and
 the cotton fibers. Cotton fiber is a differentiated single epidermal cell
 of the outer integument of the ovule. It has four distinct growth phases;
 initiation, elongation (primary cell wall synthesis), secondary cell wall
 synthesis, and maturation. Initiation of fiber development appears to be
 triggered by hormones. The primary cell wall is laid down during the
 elongation phase, lasting up to 25 days postanthesis (DPA). Synthesis of
 the secondary wall commences prior to the cessation of the elongation
 phase and continues to approximately 40 DPA, forming a wall of almost pure
 cellulose. In addition to ovary tissue promoters, transcriptional
 initiation regions from genes expressed preferentially in seed tissues,
 and in particular seed coat tissues, are also of interest for applications
 where modification of cotton fiber cells is considered.
 An example of a gene which is expressed at high levels in Brassica seed
 coat cells is the EA9 gene described in EPA 0 255 378. The nucleic acid
 sequence of a portion of the EA9 cDNA is provided therein, and can be used
 to obtain corresponding sequences, including the promoter region. An
 additional seed gene which is expressed in seed embryo and seed coat cells
 is the Bce4 Brassica gene. The promoter region from this gene also finds
 use in the subject invention; this gene and the corresponding promoter
 region are described in WO 91/13980, which was published Sep. 19, 1991.
 Fiber-specific proteins are developmentally regulated. Thus,
 transcriptional initiation regions from proteins expressed in fiber cells
 are also of interest. An example of a developmentally regulated fiber cell
 protein, is E6 (John and Crow, Proc. Natl. Acad. Sci. (1992)
 89:5769-5773). The E6 gene is most active in fiber, although low levels of
 transcripts are found in leaf, ovule and flower.
 To obtain a specifically derived transcriptional initiation region, the
 following steps may be employed. Messenger RNA (mRNA) is isolated from
 tissue of the desired developmental stage. This mRNA is then used to
 construct cDNA clones which correspond to the mRNA population both in
 terms of primary DNA sequence of the clones and in terms of abundance of
 different clones in the population. mRNA is also isolated from tissue of a
 different developmental stage in which the target gene should not be
 expressed (alternate tissue). Radioactive cDNA from the desired tissue and
 from the alternate tissue is used to screen duplicate copies of the cDNA
 clones. The preliminary screen allows for classification of the cDNA
 clones as those which correspond to mRNAs which are abundant in both
 tissues, those which correspond to mRNAs which are not abundant in either
 tissue and those which correspond to mRNAs which are abundant in one
 tissue and relatively non-abundant in the other. Clones are then selected
 which correspond to mRNAs that are abundant only in the desired tissue and
 then these selected clones are further characterized.
 Since the hybridization probe for the preliminary screen outlined above is
 total cDNA from a particular tissue, it hybridizes primarily to the most
 abundant sequences. In order to determine the actual level of expression,
 particularly in tissue where the mRNA is not as abundant, the cloned
 sequence is used as a hybridization probe to the total mRNA population(s)
 of the desired tissue(s) and various undesired tissue(s). This is most
 commonly done as a Northern blot which gives information about both the
 relative abundance of the mRNA in particular tissues and the size of the
 mRNA transcript.
 It is important to know whether the abundance of the mRNA is due to
 transcription from a single gene or whether it is the product of
 transcription from a family of genes. This can be determined by probing a
 genomic Southern blot with the cDNA clone. Total genomic DNA is digested
 with a variety of restriction enzymes and hybridized with the radioactive
 cDNA clone. From the pattern and intensity of the hybridization, one can
 distinguish between the possibilities that the mRNA is encoded either by
 one or two genes or by a large family of related genes. It can be
 difficult to determine which of several cross-hybridizing genes encodes
 the abundantly expressed mRNA found in the desired tissue. For example,
 tests indicate that pZ130 (see Example 4) is a member of a small gene
 family however, the pZ7 probe is capable of distinguishing pZ130 from the
 remainder of the family members.
 The cDNA obtained as described can be sequenced to determine the open
 reading frame (probable protein-coding region) and the direction of
 transcription so that a desired target DNA sequence later can be inserted
 at the correct site and in the correct orientation into a transcription
 cassette. Sequence information for the cDNA clone also facilitates
 characterization of corresponding genomic clones including mapping and
 subcloning as described below. At the same time, a genomic library can be
 screened for clones containing the complete gene sequence including the
 control region flanking the transcribed sequences. Genomic clones
 generally contain large segments of DNA (approximately 10-20 kb) and can
 be mapped using restriction enzymes, then subcloned and partially
 sequenced to determine which segments contain the developmentally
 regulated gene.
 Using the restriction enzyme map and sequence information, plasmids can be
 designed and constructed which have the putative ovary gene or other
 desired promoter regions attached to genes which are to be expressed in
 ovary and/or other desired tissue, particularly ovary-derived tissue.
 These hybrid constructions are tested for their pattern of expression in
 transformed, regenerated plants to be sure that the desired timing and/or
 tissue expression and/or the overall level of expression has been
 maintained successfully when the promoter is no longer associated with the
 native open reading frame. Using the method described above, several
 transcriptional regulatory regions have been identified. One example is
 the tomato derived transcriptional initiation region which regulates
 expression of the sequence corresponding to the pZ130 cDNA clone.
 Sequences hybridizable to the pZ130 clone, for example, probe pZ7, show
 abundant mRNA, especially at the early stages of anthesis. The message is
 expressed in ovary integument and ovary outer pericarp tissue and is not
 expressed, or at least is not readily detectable, in other tissues or at
 any other stage of fruit development. Thus, the pZ130 transcriptional
 initiation region is considered ovary-specific for purposes of this
 invention. FIG. 1 provides the DNA sequence of cDNA clone pZ130. The amino
 acid sequence encoded by the structural gene comprising pZ130 is
 homologous to that of a reported thionin protein (See, Qing et al., Mol.
 Gen. Genet. (1992) 234:89-96). Although thionins are reported to play a
 role in plant defense, the function of the thionin proteins, especially in
 plant ovary tissue, remains unknown.
 Downstream from, and under the regulatory control of, the ovary tissue
 transcriptional/translational initiation control region is a nucleotide
 sequence of interest which provides for modification of the phenotype of
 structures maturing from ovary tissue, such as fruit or fiber. The
 nucleotide sequence may be any open reading frame encoding a polypeptide
 of interest, for example, an enzyme, or a sequence complementary to a
 genomic sequence, where the genomic sequence may be an open reading frame,
 an intron, a noncoding leader sequence, or any other sequence where the
 complementary sequence inhibits transcription, messenger RNA processing,
 for example, splicing, or translation. The nucleotide sequences of this
 invention may be synthetic, naturally derived, or combinations thereof.
 Depending upon the nature of the DNA sequence of interest, it may be
 desirable to synthesize the sequence with plant preferred codons. The
 plant preferred codons may be determined from the codons of highest
 frequency in the proteins expressed in the largest amount in the
 particular plant species of interest. Phenotypic modification can be
 achieved by modulating production either of an endogenous transcription or
 translation product, for example as to the amount, relative distribution,
 or the like, or an exogenous transcription or translation product, for
 example to provide for a novel function or products in a transgenic host
 cell or tissue.
 Of particular interest are DNA sequences encoding expression products
 associated with regulation of plant cell growth and development,
 particularly those involved in the metabolism of hormones involved in the
 regulation of plant fruit development, such as cytokinins, auxins,
 ethylene, abscissic acid, giberrillic acid and the like. Methods and
 compositions for modulating cytokinin expression are described in U.S.
 Pat. No. 5,177,307, which disclosure is hereby incorporated by reference.
 Alternatively, various genes, from sources including other eukaryotic or
 prokaryotic cells, including bacteria, such as those from Agrobacterium
 tumefaciens T-DNA auxin and cytokinin biosynthetic gene products, for
 example, and mammals, for example interferons, may be used. Genes in the
 ethylene pathway which could be of interest include DNA sequences encoding
 ACC synthase (WO 92/04456) and pTOM13 (WO 91/01375). In fact, any gene
 coding for an enzyme involved in the ethylene biosynthesis pathway has
 potential for this application, such as SAM synthase.
 Alternatively, it is possible to introduce and express DNA sequences
 encoding enzymes capable of degrading ethylene precursors in a plant cell
 to provide ethylene resistant cells. Examples of such enzymes are ACCD
 (PCT/US91/02958) and SAM decarboxylase (WO 91/09112) Each of the
 above-cited references of this paragraph are specifically incorporated by
 reference hereunder.
 Modification of cotton fiber strength, texture or dimensional
 characteristics may utilize transcriptional cassettes when the
 transcription of an anti-sense sequence is desired. When the expression of
 a polypeptide is desired, expression cassettes providing for transcription
 and translation of the DNA sequence of interest will be used. Various
 changes are of interest; these changes may include modulation (increase or
 decrease) of formation of particular saccharides, hormones, enzymes, or
 other biological parameters. These also include modifying the composition
 of the final fruit or fiber, that is changing the ratio and/or amounts of
 water, solids, fiber or sugars. Other phenotypic properties of interest
 for modification include response to stress, organisms, herbicides,
 brushing, growth regulators, and the like. These results can be achieved
 by providing for reduction of expression of one or more endogenous
 products, particularly an enzyme or cofactor, either by producing a
 transcription product which is complementary (anti-sense) to the
 transcription product of a native gene, so as to inhibit the maturation
 and/or expression of the transcription product, or by providing for
 expression of a gene, either endogenous or exogenous, to be associated
 with the development of a plant fruit.
 The termination region which is employed in the expression cassette will be
 primarily one of convenience, since the termination regions appear to be
 relatively interchangeable. The termination region may be native with the
 transcriptional initiation region, may be native with the DNA sequence of
 interest, may be derived from another source. The termination region may
 be naturally occurring, or wholly or partially synthetic. Convenient
 termination regions are available from the Ti-plasmid of A. tumefaciens,
 such as the octopine synthase and nopaline synthase termination regions.
 In some embodiments, it may be desired to use the 3' termination region
 native to the ovary tissue transcription initiation region used in a
 particular construct.
 In some instances, it may be useful to include additional nucleotide
 sequences in the constructs to provide for targeting of a particular gene
 product to specific cell organelles. For example, where coding sequences
 for synthesis of aromatic colored pigments are used in constructs,
 particularly coding sequences enzymes which have as their substrates
 aromatic compounds such as tyrosine and indole, it is preferable to
 include sequences which provide for delivery of the enzyme into plastids,
 such as an SSU transit peptide sequence.
 For example, for melanin production the tyrosinase and ORF438 genes from
 Streptomyces antibioticus (Berman et al., (1985) 37: 101-110) are provided
 in cotton fiber cells for expression from a pZ130 promoter. In
 Streptomyces, the ORF438 and tyrosinase proteins are expressed from the
 same promoter region. For expression from constructs in a transgenic plant
 genome, the coding regions may be provided under the regulatory control of
 separate promoter regions. The promoter regions may be the same or
 different for the two genes. Alternatively, coordinate expression of the
 two genes from a single plant promoter may be desired. Constructs for
 expression of the tyrosinase and ORF438 gene products from pZ130 promoter
 regions are described in detail in the following examples. Additional
 promoters may also be desired, for example plant viral promoters, such as
 CaMV 35S, can be used for constitutive expression of one of the desired
 gene products, with the other gene product being expressed in cotton fiber
 tissues from the pZ130 promoter. In addition, the use of other plant
 promoters for expression of genes in cotton fibers is also considered,
 such as the Brassica seed promoters and the E6 gene promoter discussed
 above. Similarly, other constitutive promoters may also be useful in
 certain applications, for example the mas, Mac or DoubleMac, promoters
 described in U.S. Pat. No. 5,106,739 and by Comai et al., Plant Mol. Biol.
 (1990) 15:373-381). When plants comprising multiple gene constructs are
 desired the plants may be obtained by co-transformation with both
 constructs, or by transformation with individual constructs followed by
 plant breeding methods to obtain plants expressing both of the desired
 genes.
 The various constructs normally will be joined to a marker for selection in
 plant cells. Conveniently, the marker may be resistance to a biocide,
 particularly an antibiotic, such as kanamycin, G418, bleomycin,
 hygromycin, chloramphenicol, or the like. The particular marker employed
 will be one which will allow for selection of transformed cells as
 compared to cells lacking the DNA which has been introduced. Components of
 DNA constructs including transcription cassettes of this invention may be
 prepared from sequences which are native (endogenous) or foreign
 (exogenous) to the host. By foreign is intended that the sequence is not
 found in the wild-type host into which the construct is introduced.
 Heterologous constructs will contain at least one region which is not
 native to the gene from which the ovary tissue transcription initiation
 region is derived.
 In preparing the constructs, the various DNA fragments may be manipulated,
 so as to provide for DNA sequences in the proper orientation and, as
 appropriate, in proper reading frame for expression; adapters or linkers
 may be employed for joining the DNA fragments or other manipulations may
 be involved to provide for convenient restriction sites, removal of
 superfluous DNA, removal of restriction sites, or the like. In vitro
 mutagenesis, primer repair, restriction, annealing, resection, ligation,
 or the like may be employed, where insertions, deletions or substitutions,
 e.g. transitions and transversions, may be involved. Conveniently, a
 vector or cassette may include a multiple cloning site downstream from the
 ovary-related transcription initiation region, so that the construct may
 be employed for a variety of sequences in an efficient manner.
 In carrying out the various steps, cloning is employed, so as to amplify
 the amount of DNA and to allow for analyzing the DNA to ensure that the
 operations have occurred in proper manner. By appropriate manipulations,
 such as restriction, chewing back or filling in overhangs to provide blunt
 ends, ligation of linkers, or the like, complementary ends of the
 fragments can be provided for joining and ligation. A wide variety of
 cloning vectors are available, where the cloning vector includes a
 replication system functional in E. coli and a marker which allows for
 selection of the transformed cell. Illustrative vectors include pBR322,
 pUC series, M13mp series, pACYC184, etc. Thus, the sequence may be
 inserted into the vector at an appropriate restriction site(s), the
 resulting plasmid used to transform the E. coli host, the E. coli grown in
 an appropriate nutrient medium and the cells harvested and lysed and the
 plasmid recovered. Analysis may involve sequence analysis, restriction
 analysis, electrophoresis, or the like. After each manipulation the DNA
 sequence to be used in the final construct may be restricted and joined to
 the next sequence. Each of the partial constructs may be cloned in the
 same or different plasmids.
 A variety of techniques are available and known to those skilled in the art
 for introduction of constructs into a plant cell host. These techniques
 include transfection with DNA employing A. tumefaciens or A. rhizogenes as
 the transfecting agent, protoplast fusion, injection, electroporation,
 particle acceleration, etc. For transformation with Agrobacterium,
 plasmids can be prepared in E. coli which contain DNA homologous with the
 Ti-plasmid, particularly T-DNA. The plasmid may or may not be capable of
 replication in Agrobacterium, that is, it may or may not have a broad
 spectrum prokaryotic replication system such as does, for example, pRK290,
 depending in part upon whether the transcription cassette is to be
 integrated into the Ti-plasmid or to be retained on an independent
 plasmid. The Agrobacterium host will contain a plasmid having the vir
 genes necessary for transfer of the T-DNA to the plant cell and may or may
 not have the complete T-DNA. At least the right border and frequently both
 the right and left borders of the T-DNA of the Ti- or Ri-plasmids will be
 joined as flanking regions to the transcription construct. The use of
 T-DNA for transformation of plant cells has received extensive study and
 is amply described in EPA Serial No. 120,516, Hoekema, In: The Binary
 Plant Vector System Offset-drukkerij, Kanters B. V., Alblasserdam, 1985,
 Chapter V, Knauf, et al., Genetic Analysis of Host Range Expression by
 Agrobacterium, In: Molecular Genetics of the Bacteria-Plant Interaction
 Puhler, A. ed., Springer-Verlag, NY, 1983, p. 245, and An, et al., EMBO J.
 (1985) 4:277-284.
 For infection, particle acceleration and electroporation, a disarmed Ti
 plasmid lacking the tumor genes found in the T-DNA region may be
 introduced into the plant cell. By means of a helper plasmid, the
 construct may be transferred to the A. tumefaciens and the resulting
 transfected organism used for transfecting a plant cell; explants may be
 cultivated with transformed A. tumefaciens or A. rhizogenes to allow for
 transfer of the transcription cassette to the plant cells. Alternatively,
 to enhance integration into the plant genome, terminal repeats of
 transposons may be used as borders in conjunction with a transposase. In
 this situation, expression of the transposase should be inducible, so that
 once the transcription construct is integrated into the genome, it should
 be relatively stably integrated. Transgenic plant cells are then placed in
 an appropriate selective medium for selection of transgenic cells which
 are then grown to callus, shoots grown and plantlets generated from the
 shoot by growing in rooting medium.
 To confirm the presence of the transgenes in transgenic cells and plants, a
 Southern blot analysis can be performed using methods known to those
 skilled in the art. Expression products of the transgenes can be detected
 in any of a variety of ways, depending upon the nature of the product, and
 include immune assay, enzyme assay or visual inspection, for example to
 detect pigment formation in the appropriate plant part or cells. Once
 transgenic plants have been obtained, they may be grown to produce fruit
 having the desired phenotype. The fruit or fruit parts, such as cotton
 fibers may be harvested, and/or the seed collected. The seed may serve as
 a source for growing additional plants having the desired characteristics.
 The terms transgenic plants and transgenic cells include plants and cells
 derived from either transgenic plants or transgenic cells.
 The various sequences provided herein may be used as molecular probes for
 the isolation of other sequences which may be useful in the present
 invention, for example, to obtain related transcriptional initiation
 regions from the same or different plant sources. Related transcriptional
 initiation regions obtainable from the sequences provided in this
 invention will show at least about 60% homology, and more preferred
 regions will demonstrate an even greater percentage of homology with the
 probes. Of particular importance is the ability to obtain related
 transcription initiation control regions having the timing and tissue
 parameters described herein. For example, using the probe pZ130 at least 7
 additional clones have been identified, but not further characterized.
 Thus, by employing the techniques described in this application, and other
 techniques known in the art (such as Maniatis, et al., Molecular Cloning,
 A Laboratory Manual (Cold Spring Harbor, N.Y.) 1982), other transcription
 initiation regions capable of directing ovary tissue transcription as
 described in this invention may be determined. The constructs can also be
 used in conjunction with plant regeneration systems to obtain plant cells
 and plants; the constructs may also be used to modify the phenotype of a
 fruit and fruits produced thereby.
 For cotton applications, various varieties and lines of cotton may find use
 in the described methods. Cultivated cotton species include Gossypium
 hirsutum and G. babadense (extra-long staple, or Pima cotton), which
 evolved in the New World, and the Old World crops G. herbaceum and G.
 arboreum.
 The following examples are offered by way of illustration and not by
 limitation.
 Experimental
 The following deposits have been made at the American Type Culture
 Collection (ATCC) (12301 Parklawn Drive, Rockville, Md. 20852).
 Bacteriophage Calgene Lambda 116 and Calgene Lambda 140, containing the
 genomic sequence of pZ70 and pZ130, respectively, were deposited on Jul.
 13, 1989 and were given accession numbers 40632 and 40631, respectively.
 EXAMPLE 1
 Construction of Pre-Anthesis Tomato Ovary cDNA Banks and Screening for
 Ovary-Specific Clones
 cDNA Library Preparation
 Tomato plants (Lycopersicon esculentum cv UC82B) were grown under
 greenhouse conditions. Poly(A)+RNA was isolated as described by Mansson et
 al., Mol. Gen. Genet. (1985) 200:356-361. The synthesis of cDNA from
 poly(A)+RNA, prepared from ovaries of unopened tomato flowers
 (pre-anthesis stage), was carried out using the BRL cDNA Cloning Kit
 following the manufacturer's instructions (BRL; Bethesda, Md.). Addition
 of restriction endonuclease EcoRI linkers (1078, New England Biolabs;
 Beverly, Mass.) to the resulting double-stranded cDNA was accomplished by
 using the procedures described in Chapter 2 of DNA Cloning Vol. 1: A
 Practical Approach, Glover, ed., (IRL Press, Oxford 1985). Cloning the
 cDNA into the EcoRI site of the phage Lambda ZAP (Stratagene; La Jolla,
 Calif.) and packaging the resulting recombinant phage (using GigaPack
 Gold, Stratagene) was carried out as described in the respective
 commercial protocols.
 Two cDNA libraries were prepared as described above from the same
 pre-anthesis stage mRNA. For the second library, which contained
 significantly longer cDNA than the first, the poly(A)+RNA sample was run
 through an RNA spin column (Boehringer Mannheim Biochemicals;
 Indianapolis, Ind.), following the manufacturer's directions, prior to the
 cloning procedures.
 cDNA Library Screening
 The first cDNA library was screened by differential hybridization using
 32P-labeled cDNA probes made from pre-anthesis mRNA, leaf mRNA and young
 seedling mRNA. Clones were selected based on hybridization to only
 preanthesis mRNA. The cDNAs corresponding to the selected Lambda ZAP
 (Stratagene) clones were excised from the phage vector and propagated as
 plasmids (following the manufacturer's instructions).
 From an initial screen of 1000 cDNAs, 30 selected clones falling into five
 classes based on the sequences of their cDNA inserts were isolated. Two
 clones, clones pZ7 and pZ8, were selected for further study. The DNA
 sequences of pZ7 and pZ8 are shown as the underlined portions of FIGS. 1
 and 4, respectively.
 Several thousand recombinant clones from the second cDNA library were
 screened by plaque hybridization (as described in the Stratagene Cloning
 Kit Instruction Manual) with a mixture of radiolabeled DNA probes.
 Screening of approximately three thousand recombinant clones from the
 second library with the pZ7 and pZ8 DNA probes yielded selection of
 fourteen clones which had intense hybridization signals. The clones
 selected were excised from the phage vector and propagated as plasmids.
 DNA was isolated from each clone, cut with the restriction endonuclease
 EcoRI, then electrophoresed through a 0.7% agarose gel. Duplicate blot
 hybridizations were performed as described in Maniatis et al., Molecular
 Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y., 1982) with
 radiolabeled probes representing the genes of interest (pZ7 and pZ8).
 Seven clones which hybridized to pZ7 and three clones which hybridized to
 pZ8 were selected. The longest of these for each probe, pZ130
 (pZ7-hybridizing) and pZ70 (pz8hybridizing), were characterized further
 and used in additional experiments.
 EXAMPLE 2
 Analysis of cDNA Clones
 Northern Analysis
 Tissue-specificity of the cDNA clones was demonstrated as follows: RNA was
 isolated from 1, 2, 3, 4, 5, 6, 7, 10, 14, 17 and 21 day post-anthesis,
 anthesis and pre-anthesis stage tomato ovaries, tomato leaves and
 unorganized tomato callus using the method of Ecker and Davis (Proc. Natl.
 Acad. Sci. (1987) 84:5203) with the following modifications. After the
 first precipitation of the nucleic acid, the pellets were resuspended in 2
 ml of diethylpyrocarbonate (DEP)treated water on ice. The solutions were
 brought to 1 mM MgC12 and 1/4 volume of 8 M LiCl was added. The samples
 were mixed well and stored at 4.degree. C. overnight. The samples were
 then centrifuged at 8,000 RPM for 20 min. at 4.degree. C. The pellets were
 dried, resuspended in DEP-treated water on ice as before and
 ethanol-precipitated once more. The RNAs were electrophoresed on
 formaldehyde/agarose gels according to the method described by Fourney et
 al., Focus (1988) 10:5-7, immobilized on Nytran membranes (Schleicher &
 Schuell; Keene, N.H.) and hybridized with 32P-labeled probes.
 Based upon the Northern analysis with a 32P-labeled pZ7 EcoRI insert DNA or
 a pZ8 EcoRI insert DNA, it is clear that both of these genes are most
 highly expressed at anthesis in tomato variety UC82B and somewhat less
 highly expressed prior to and a day following the opening of the flower.
 FIG. 6 shows tomato flowers at various stages of development and
 immediately below, a representative ovary dissected from a flower at the
 same stage of development. As seen in FIG. 6, by two days after the onset
 of anthesis, the expression of both genes had dropped off dramatically.
 The size of the mRNA species hybridizing to the pZ7 probe was
 approximately 800 nt and to the pZ8 probe approximately 500 nt.
 From two days post-anthesis, pZ8 RNA accumulation was apparently maintained
 at a relatively low level while pZ7 RNA accumulation continued to drop off
 steadily until, by three weeks post-anthesis, it was undetectable by this
 analysis. pZ8 RNA accumulation was not detectable by the method described
 above in RNA samples isolated from tomato fruit older than the immature
 green stage of fruit ripening. No RNA hybridizing to pZ7 or pZ8 was found
 in callus tissue; no RNA hybridizing to pZ7 was found in leaf tissue; on
 longer exposures a barely detectable hybridization signal for pZ8 was seen
 in leaf RNA.
 Expression Level
 Message abundance corresponding to the cDNA probes was determined by
 comparing the hybridization intensity of a known amount of RNA synthesized
 in vitro from the clones (using T7 or T3 RNA polymerase in the Riboprobe
 System (Promega)) to RNA from anthesis stage and three week old tomato
 ovaries. This analysis indicated that pZ7 and pZ8 cDNAs represent abundant
 RNA classes in anthesis-stage tomato ovaries, being approximately 5% and
 2% of the message, respectively.
 Cellular Specificity
 The cellular specificity of the cDNA probes may be demonstrated using the
 technique of in situ hybridization. Preanthesis stage UC82B tomato ovaries
 were fixed overnight in a 4% paraformaldehyde, phosphate buffered saline
 (PBS), 5 mM MgCl2 solution, pH 7.4 (PBS is 10 mM phosphate buffer, pH 7.4,
 150 mM NaCl) (Singer et al., Biotechniques (1986) 4:230-250). After
 fixation, the tissue was passed through a graded tertiary butyl alcohol
 (TBA) series, starting at 50% alcohol, infiltrated with Paraplast and cast
 into paraffin blocks for sectioning (Berlyn and Miksche, Botanical
 Microtechnique and Cytochemistry, (1976) Iowa). Embedded ovaries were
 transversely cut, 8 .mu.m thick sections, on a Reichert Histostat rotary
 microtome. Paraffin ribbons holding 5-7 ovary sections were affixed to
 gelatin-chrom alum subbed slides (Berlyn and Miksche (1976) supra) and
 held in a dust-free box until in situ hybridizations were performed.
 Slides ready to be hybridized were deparaffinized in xylene and rehydrated
 by passing through an ethanol hydration series as described in Singer et
 al., supra (1986).
 A 2.times.hybridization mix was made consisting of 100 .mu.l 20.times.SSC,
 20 .mu.l 10% BSA, 100 111 750 mM DTT, 200 .mu.l 50% dextran sulfate, 50
 .mu.l RNasin, and 30 .mu.l sterile water. Sense and antisense .sup.35
 S-RNA probes were generated from cDNAs of interest using T3 and T7 RNA
 polymerases in vitro transcription (Riboprobe Promega Biotec or
 Stratagene) reactions following the manufacturer's protocol. 2.5 .mu.l
 tRNA (20 mg/ml), 2.5 .mu.l salmon sperm DNA (10 mg per ml) and 4.times.106
 cpm/ probe were dried down using a lyophilizer. This mix was then
 resuspended in 25 .mu.l 90% formamide containing 25 .mu.l
 2.times.hybridization mix per slide. 40 .mu.l of this hybridization mix
 was placed on each slide. A cover slip was placed over the sections and
 edges sealed with rubber cement. Slides were placed in slide holders
 inside a glass slide box, covered, and placed in a 37.degree. C. dry oven
 overnight to hybridize. Posthybridization treatments were as described in
 Singer et al., (1986), supra.
 Autoradiography was performed as described in KODAK Materials for Light
 Microscope (KODAK (1986); Rochester, N.Y.) using liquid emulsion NTB-3.
 Slides are left to expose in a light-tight box for approximately two
 weeks. After developing the autoradiographic slides, sections were stained
 in 0.05% toluidine blue and then dehydrated through a graded alcohol
 series; xylene:100% ethanol, 1:1, followed by 2 changes of 100% xylene,
 five minutes in each solution. Coverslips were mounted with Cytoseal (VWR;
 San Francisco, Calif.) and left on a slide warmer until dry (45-50.degree.
 C., 1-2 days). Autoradiographic slides were then ready for microscopic
 examination.
 When pre-anthesis tomato ovaries were hybridized to sense and antisense
 .sup.35 S-pZ7 RNA, the antisense transcripts hybridized specifically to
 the outer pericarp region of the ovary and to the outer region of the
 ovules (the integuments). The sense transcripts (negative control) showed
 no hybridization. When preanthesis tomato ovaries were hybridized to sense
 and antisense .sup.35 S-pZ8 RNA, the antisense transcript hybridized
 specifically to the inner core region of the ovary and to the outer region
 of the ovules. The sense transcripts showed no hybridization.
 In summary, the mRNA transcripts encoded by the genes corresponding to pZ7
 and pZ8 were abundantly expressed during a very specific stage of tomato
 fruit development, primarily at anthesis and at a day prior to and after
 the opening of the flower. The transcripts additionally were expressed in
 a specific subset of tomato ovary cell types during that stage of
 development particularly in the integuments (pZ7 and pZ8) as well as the
 ovarian outer pericarp (pZ7) and inner core region (pZ8).
 EXAMPLE 3
 Sequencing of pZ130 and pZ70 cDNA Clones
 The complete DNA sequences of the cDNA pZ130 and pZ70 clones were
 determined using the Sanger et al., (1971) dideoxy technique. The DNA
 sequences of both pZ130 and pZ70 were translated in three frames. The
 sequences, including the longest open reading frame for each, are shown in
 FIG. 1 (pZ130) and FIG. 4 (pZ70).
 EXAMPLE 4
 Analysis of Gene Family
 Southern analysis was performed as described by Maniatis et al., supra,
 (1982). Total tomato DNA from cultivar UC82B was digested with BamHI,
 EcoRI and HindIII, separated by agarose gel electrophoresis and
 transferred to nitrocellulose. Southern hybridization was performed using
 32P-labeled probes produced by random priming of pZ130 or pZ70. A simple
 hybridization pattern indicated that the genes encoding pZ130 and pZ70 are
 present in a few or perhaps only one copy in the tomato genome.
 Additional analysis, using a pZ130 hybridization probe to hybridize to
 tomato genomic DNA digested with the restriction endonuclease BglII,
 indicated that this gene is actually a member of a small (approximately
 5-7 member) family of genes. The original pZ7 cDNA clone, consisting of
 sequences restricted to the 3' untranslated region of the longer pZ130
 clone, however, hybridizes intensely only to one band and perhaps faintly
 to a second band based on Southern analysis using BglII digested tomato
 genomic DNA.
 EXAMPLE 5
 Preparation of Genomic Clones pZ130 and pZ70
 Two genomic clones, one representing each of cDNA clones pZ130 and pZ70,
 were obtained as follows. A genomic library constructed from DNA of the
 tomato cultivar UC82B, partially digested with the restriction
 endonuclease Sau3A, was established in the lambda phage vector, lambda-FIX
 according to the manufacturer's instructions (Stratagene; La Jolla,
 Calif.). This library was screened using 32P-labeled pZ130 and pZ70 as
 probes. A genomic clone containing approximately 14.5 kb of sequence from
 the tomato genome which hybridized to pZ70 was isolated. The region which
 hybridizes to the pZ70 probe was found within the approximately 2 kb
 XbaI-HindIII restriction fragment of Calgene Lambda 116 (See FIG. 5). A
 second genomic clone, containing approximately 13 kb of sequence from the
 tomato genome and hybridizing to pZ130 (and pZ7) was isolated. The region
 which hybridized to the pZ130 probe was found within the larger EcoRI
 HindIII restriction fragment of Calgene Lambda 140 (See FIG. 3).
 Preparation of pCGN2015
 pCGN2015 was prepared by digesting pCGN565 with XbaI, blunting with mung
 bean nuclease, and inserting the resulting fragment into an EcoRV digested
 BluescriptKSM13-(Stratagene) vector to create pCGN2008. pCGN2008 was
 digested with EcoRI and HindIII, blunted with Klenow, and the 1156 bp
 chloramphenicol fragment isolated. BluescriptKSM13+ (Stratagene) was
 digested with DraI and the 2273 bp fragment isolated and ligated with the
 pCGN2008 chloramphenicol fragment creating pCGN2015.
 Preparation of pCGN2901/pCGN2902
 pCGN2901 contains the region surrounding the pZ7-hybridizing region of the
 pZ130 genomic clone, including approximately 1.8 kb in the 5' direction
 and approximately 4 kb in the 3'-direction. To prepare pCGN2901, Calgene
 Lambda 140 was digested with SalI and the resulting fragment which
 contains the pZ7-hybridizing region was inserted into pCGN2015, at the
 pCGN2015 unique SalI site, to create pCGN2901.
 pCGN2902 contains the other SalI fragment (non-pZ7-hybridizing) of the
 pZ130 genome derived from SalI digestion of Calgene Lambda 140, also put
 into a pCGN2015 construct.
 EXAMPLE 6
 Preparation of a pZ130 Expression Construct
 Plasmid DNA isolated from pCGN2901 was digested to completion with NcoI and
 then treated with exonuclease isolated from mung bean (Promega, Madison,
 Wis.) to eliminate single-stranded DNA sequences including the ATG
 sequence making up a portion of the NcoI recognition sequence. The sample
 was then digested to completion with SacI. The resulting 1.8 kb
 (approximate) 5' SacI to NcoI fragment was then inserted into a
 pUC-derived ampicillin-resistant plasmid, pCGP261 (described below), that
 had been prepared as follows. pCGP261 was digested to completion with
 XbaI, the single-stranded DNA sequences were filled in by treatment with
 the Klenow fragment of DNA polymerase I, and the pCGP261 DNA redigested
 with SacI. The resulting expression construct, designated pCGN2903,
 contained, in the 5' to 3' direction of transcription, an ovary tissue
 promoter derived from Lambda 140, a tmr gene and tmr 3'-transcriptional
 termination region.
 The plasmid pCGP261 contains the sequences from position 8,762 through
 9,836 from the Agrobacterium tumefaciens octopine Ti plasmid pTilS955 (as
 sequenced by Barker et al., Plant Mol. Biol. (1983) 2:335-350). This
 region contains the entire coding region for the genetic locus designated
 tmr which encodes isopentenyltransferase (Akiyoshi et al., Pro. Natl.
 Acad. Sci. (1984) 81:4776-4780), 8 bp 5' of the translation initiation ATG
 codon and 341 bp of sequences 3' to the translation stop TAG codon.
 Plasmid pCGP261 was created as follows. Plasmid pCGN1278 (described in U.S.
 Pat. No. 5,177,307, filed Jul. 127, 1990, which is hereby incorporated in
 its entirety by reference) was digested with XbaI and EcoRI. The
 single-stranded DNA sequences produced were filled in by treatment with
 the Klenow fragment of DNA polymerase I. The XbaI to EcoRI fragment
 containing the tmr gene was then ligated into the vector ml3 Bluescript
 minus (Stratagene Inc., La Jolla, Calif.) at the SmaI site, resulting in
 plasmid pCGP259. All of the region found upstream of the ATG translation
 initiation codon and some of the tmr gene coding region was eliminated by
 digesting pCGP259 with BSpMI and BstXI. The resulting coding region and 8
 bp of the sequence originally found upstream of the first ATG codon was
 re-introduced into the plasmid and an XbaI site introduced into the
 plasmid via a synthetic oligonucleotide comprising the following sequence:
 5' AATTAGATGCAGGTCCATAAGTTTTTTCTAGACGCG 3' (SEQ ID NO: 4). The resulting
 plasmid is pCGP261.
 An XbaI to KpnI fragment of pCGN2903 containing the pZ130 gene 5' and tmr
 gene coding and 3' region construct was then inserted into the binary
 cassette pCGN1557, and designated pCGN2905. Transgenic plants were
 prepared. (See U.S. Pat. No. 5,177,307, described above).
 EXAMPLE 7
 Preparation of pZ130 Promoter Cassette
 The pZ130 cassette contains 1.8 kb (pCGN2909) or 5 kb (pCGN2928) of DNA 5'
 of the translational start site and the 3' region (from the TAA stop codon
 to a site 1.2 kb downstream) of the pZ130 gene. The pZ130 cassettes were
 constructed as follows.
 Transcriptional Initiation Region
 Plasmid DNA isolated from pCGN2901 (see U.S. Pat. No. 5,177,307, above) was
 digested to completion with NcoI and then treated with exonuclease
 isolated from mung bean (Promega, Madison, Wis.) to eliminate
 single-stranded DNA sequences, including the ATG sequence making up a
 portion of the NcoI recognition sequence. The sample was then digested to
 completion with SacI. The resulting 1.8 kb 5' SacI to NcoI fragment was
 then inserted into pCGN2015 (described above) to create pCGN2904.
 In order to eliminate redundant restriction enzyme sites and make
 subsequent cloning easier, plasmid DNA isolated from pCGN2904 was digested
 to completion with SalI and EcoRI and the resulting 1.8 kb fragment,
 containing the pZ130 5' sequences, inserted into pBluescriptII
 (Stratagene; La Jolla, Calif.) to create pCGN2907.
 Transcriptional and Translational Termination Region
 Plasmid DNA isolated from pCGN2901 was digested to completion with EcoRI
 and BamHI. The resulting 0.72 kb EcoRI to BamHI fragment located
 downstream (3') from the pZ130 coding region was inserted into pCGN2907
 creating pCGN2908.
 The insertion of the 0.5 kb (approximately) DNA sequence, including the
 pZ130 gene TAA stop codon and those sequences between the stop codon and
 the EcoRI site downstream (3') and the addition of unique restriction
 sites to facilitate insertion of foreign genes, was accomplished as
 follows.
 A polylinker/"primer" comprising the sequence
 5'GTTCCTGCAGCATGCCCGGGATCGATAATAATTAAGTGAGGC-3' (SEQ ID NO:5) was
 synthesized to create a polylinker with the following sites:
 PstI-SphI-SmaI-ClaI and to include the pZ130 gene TAA stop codon and the
 following (3') 13 base pairs of the pZ130 gene 3' region sequence. Another
 oligonucleotide comprising the sequence 5'-CAAGAATTCATAATATTATATATAC 3'
 (SEQ ID NO:4) was synthesized to create a "primer" with an EcoRI
 restriction site and 16 base pairs of the pZ130 gene 3' region immediately
 adjacent to the EcoRI site located approximately 0.5 kb 3' of the pZ130
 gene TAA stop codon.
 These synthetic oligonucleotides were used in a polymerase chain reaction
 (PCR) in which plasmid DNA isolated from pCGN2901 was used as the
 substrate in a thermal cycler (Perkin-Elmer/Cetus, Norwalk, Conn.) as per
 the manufacturer's instructions. The resulting 0.5 kb DNA product was
 digested to completion with PstI and EcoRI and the resulting 0.5 kb DNA
 fragment inserted into pCGN2908 to create pCGN2909. The complete DNA
 sequence of the 0.5 kb region from the PstI site to the EcoRI site was
 determined using the Sanger et al., (1971) dideoxy technique to verify
 that no mistakes in the sequence had occurred between the oligonucleotide
 primers during the PCR reaction.
 The pZ130 cassette, pCGN2909, thus comprises the 5' pZ130 DNA sequences
 from the SalI site at position 808 to position 2636 (see FIG. 2), unique
 PstI, SphI and SmaI sites which can be conveniently used to insert genes,
 and the 3' pZ130 DNA sequences from the TAA stop codon at position 3173
 (FIG. 2) through the BamHI site at position 4380.
 A pZ130 cassette, pCGN2928, was prepared by inserting the 3.2 KpnI to SalI
 fragment of pCGN2059 into the KpnI and SalI sites of pCGN2909. pCGN2059
 was prepared by inserting the 3.2 SalI to BglII fragment of pCGN2902 into
 M13mpl9. pCGN2928 is thus identical to pCGN2909 except that it includes an
 additional approximately 3.2 kb of pZ130 DNA sequence upstream of the SalI
 site located at position 808 of FIG. 2.
 EXAMPLE 8
 Preparation and Analysis of Test Constructs
 A .beta.-glucuronidase (GUS) reporter gene was used to evaluate the
 expression and tissue specificity of the pZ130-GUS constructions. GUS is a
 useful reporter gene in plant systems because it produces a highly stable
 enzyme, there is little or no background (endogenous) enzyme activity in
 plant tissues, and the enzyme is easily assayed using fluorescent or
 spectrophotometric substrates. (See, for example, Jefferson, Plant Mol.
 Rep. (1987) 5:387-405.) Histochemical stains for GUS enzyme activity are
 also available which can be used to analyze the pattern of enzyme
 accumulation in transgenic plants. Jefferson (1987), supra.
 Preparation of Test Constructs pCGN2917 and pCGN2918
 These constructs contain 1.8 kb of pZ130 5' sequence, the GUS gene coding
 region and 1.2 kb of pZ130 3' sequence. pCGN2917 and pCGN2918 differ from
 each other only in the orientation of the pZ130/GUS construction with
 respect to the other elements of the binary vector plasmid for example,
 the 35S promoter from CaMV.
 The constructs were made by inserting the PstI fragment of pRAJ250
 (Jefferson (1987) supra), or any other plasmid construct having the PstI
 fragment containing the GUS coding region, into the PstI site of pCGN2909.
 The resulting plasmid, having the GUS gene in the sense orientation with
 respect to the pZ130 gene promoter region, was named pCGN2914. The
 pZ130/GUS construction was excised as an XbaI to KpnI fragment and cloned
 into the binary vectors pCGN1557 and pCGN1558 to make pCGN2917 and
 pCGN2918, respectively. pCGN1557 and pCGN1558 are described in McBride and
 Summerfelt, Plant Mol. Biol. (1990) 14:269-296.
 Preparation of Test Construct pCGN2926
 This construct contains 5 kb of pZ130 5' sequence, the GUS gene coding
 region and 1.2 kb of pZ130 3' sequence. It was made by inserting the 3.2
 kb KpnI to SalI fragment of pCGN2059 into the KpnI and SalI sites of
 pCGN2914. The resulting plasmid was named pCGN2923. The pZ130/GUS/pZ130
 construction was then excised from pCGN2923 as an XbaI to KpnI fragment
 and cloned into the binary vector pCGN1557 resulting in pCGN2926.
 Analysis of GUS Enzyme Activity
 .beta.-glucuronidase activity of transformants was measured using
 4methyl-umbelliferyl glucuronide as a substrate, as outlined in Jefferson
 (1987) supra GUS enzyme activity was easily detected in the ovaries of the
 transformed plants and quantitatively was quite high in comparison with
 the activity background observed in ovaries isolated from nontransformed
 tomato plants and from leaves of transformed plants. Interestingly, upon
 comparison of the pCGN2917 and pCGN2918 transformants, it was found that
 proximity to a 35S CaMV enhancer region (pCGN1558) may reduce, or
 eliminate, ovary-tissue specificity.
 EXAMPLE 9
 pZ7 Cotton Transformation
 Explant Preparation
 Coker 315 seeds were surface disinfected by placing in 50% Clorox.RTM.
 (2.5% sodium hypochlorite solution) for 20 minutes and rinsing 3 times in
 sterile distilled water. Following surface sterilization, seeds were
 germinated in 25.times.150 sterile tubes containing 25 mls 1/2.times.MS
 salts: 1/2.times.B5 vitamins: 1.5% glucose: 0.3% gelrite. Seedlings were
 germinated in the dark at 28.degree. C. for 7 days. On the seventh day
 seedlings were placed in the light at 28+2.degree. C.
 Cocultivation and Plant Regeneration
 Single colonies of A. tumefaciens strain 2760 containing binary plasmids
 pCGN2917 and pCGN2926 were transferred to 5 ml of mg/L broth and grown
 overnight at 30.degree. C. Bacteria cultures were diluted to 1.times.108
 cells/ml with mg/L just prior to cocultivation. Hypocotyls were excised
 from eight day old seedlings, cut into 0.5-0.7 cm sections and placed onto
 tobacco feeder plates (Horsch et al., (1985)). Feeder plates were prepared
 one day before use by plating 1.0 ml tobacco suspension culture onto a
 petri plate containing Callus Initiation Medium (CIM) without antibiotics
 (MS salts: B5 vitamins: 3% glucose: 0.1 mg/L 2,4-D: 0.1 mg/L kinetin: 0.3%
 gelrite, pH adjusted to 5.8 prior to autoclaving). A sterile filter paper
 disc (Whatman #1) was placed on top of the feeder cells prior to use.
 After all sections were prepared, each section was dipped into an A.
 tumefaciens culture, blotted on sterile paper towels and returned to the
 tobacco feeder plates.
 Following two days of cocultivation on the feeder plates, hypocotyl
 sections were placed on fresh CIM containing 75 mg/L kanamycin and 500
 mg/L carbenicillin. Tissue was incubated at 28+2.degree. C., 30 uE 16:8
 light:dark period for 4 weeks. At four weeks the entire explant was
 transferred to fresh CIM containing antibiotics. After two weeks on the
 second pass, the callus was removed from the explants and split between
 CIM and Regeneration Medium (MS salts: 40 mM KN03: 10 mM NH4Cl:B5
 vitamins:3% glucose:0.3% gelrite:400 mg/L carb:75 mg/L kanamycin).
 Embryogenic callus was identified 2-6 months following initiation and was
 subcultured onto fresh regeneration medium. Embryos were selected for
 germination, placed in static liquid Embryo Pulsing Medium (Stewart and
 Hsu medium: 0.01 mg/l NAA: 0.01 mg/L kinetin: 0.2 mg/L GA3) and incubated
 overnight at 30.degree. C. The embryos were blotted on paper towels and
 placed into Magenta boxes containing 40 mls of Stewart and Hsu medium
 solidified with Gelrite.TM.. Germinating embryos were maintained at
 28.+-.2.degree. C., 50 uEm.sup.-2 s.sup.-1, 16:8 photoperiod. Rooted
 plantlets were established in soil and transferred to the greenhouse.
 Cotton growth conditions in growth chambers are as follows: 16 hour
 photoperiod, temperature of approximately 80-85.degree., light intensity
 of approximately 500 .mu.Einsteins. Cotton growth conditions in
 greenhouses are as follows: 14-16 hour photoperiod with light intensity of
 at least 400 .mu.Einsteins, day temperature 90-95.degree. F., night
 temperature 70-75.degree. F., relative humidity to approximately 80%.
 Plant Analysis
 Flowers from greenhouse grown T1 plants were tagged at anthesis in the
 greenhouse. Squares (cotton flower buds), flowers, bolls etc. were
 harvested from these plants at various stages of development and assayed
 for GUS activity. GUS fluorometric and histochemical assays were performed
 on hand cut sections as described in Jefferson (1987), supra.
 At least ten events (transgenic plants) from each construct (pCGN2917 and
 pCGN2926) were sent to the Growth Chambers/Greenhouse. Approximately 80%
 (9/11) of the 2917 plants and 100% (12/12) of the 2926 plants expressed
 GUS at a level detectable by either fluorometric or histochemical assay.
 Squares from several of pCGN2917 and pCGN2926 transfected plants were
 assayed for GUS expression using histochemical analysis wherein the cells
 which are expressing GUS stain blue. Preliminary analysis indicates that
 all plants expressed GUS in the developing floral parts. Ovules and
 anthers stained extremely dark. Bracts and locule walls were also blue in
 some cases. Fibers from 5, 9 and 12 DPA bolls off these plants were also
 expressing GUS.
 Several GUS assays were done on developing bolls at stages from squaring
 through 53 days post anthesis. GUS activity is very high in squares and
 flowers. Activity in bolls varies from plant to plant. Activity was
 present in fiber from two of the 2926 plants at 43 and 53 dpa.
 .beta.-glucuronidase is a very stable enzyme; therefore, presence of GUS
 activity may not be directly correlated in a temporal manner with gene
 expression, however, the specificity of expression in tissues and/or
 structures derived from ovary integument was significant. Differences in
 the breakdown of GUS as well as differences in expression may explain the
 variability of expression patterns.
 Comparisons Between Cotton and Tomato GUS Expression
 An initial MUG assay was done on tissues from tomato and cotton plants
 transfected with pCGN2917. GUS activity was found in tomato roots, stems
 and leaves as well as meristems, and floral parts. The amount of activity
 varied from plant to plant. In cotton, activity was highest in floral
 parts but was detectable in roots and stems of some plants.
 Cotton Transformation
 The temporal pattern of expression of the chimeric pZ130/GUS gene in fiber
 cells of a cotton plant transformed with pCGN2926 was examined by
 isolating RNA from 7, 17-21, and 28 day post-anthesis fibers of plant
 2926-13 using the method of Hall et al., (Proc. Natl. Acad. Sci. (1978)
 75:3196) with the following modifications. After the second 2M LiCl wash,
 the pellet was dissolved in 1/10 original volume of 10 mM Tris pH7.5 and
 brought to 35 mM potassium acetate pH6.5 and 1/2 volume EtOH was added
 slowly. The mixture was placed on ice for 15 minutes and then centrifuged
 at 20,000.times.g for 15 minutes at 4.degree. C. The potassium acetate
 concentration was brought to 0.2M, 21/2 volumes EtOH added and the RNA
 placed at -20.degree. C. for several hours. The precipitate was
 centrifuged at 12,000.times.g for 30 minutes at 4.degree. C. and the
 pellet was resuspended in diethylpyrocarbonate-treated water.
 RNA was isolated from anthesis stage ovules of plant 2926-13 using the
 method described above in Example 2 with the following modification. The
 obvious precipitant present during the final ethanol-precipitation was
 carefully avoided by decanting or otherwise separating it from the
 ethanol-soluble material prior to centrifugation. The fiber and ovule RNAs
 were then processed for Northern analysis as described above in Example 2.
 Based upon Northern analysis with a .sup.32 p-labeled GUS coding region
 probe, it is clear that the chimeric pZ130/GUS gene is expressed in
 anthesis stage ovules in plant 2926-13 as evidenced by accumulation of GUS
 mRNA in those tissues. FIG. 8 provides a comparison of anthesis stage RNA
 with RNA from fibers 7, 21 and 28 days post anthesis. As seen in FIG. 8,
 by seven days after the onset of anthesis, the expression of the gene had
 dropped off dramatically in isolated fibers to levels undetectable by this
 method. This pattern of expression closely parallels the pattern observed
 for the endogenous pZ130 (thionin) gene in tomato ovaries (see FIG. 6).
 Lane A is anthesis stage ovules; lane B is 7 day old fibers; lane C is 21
 day old fibers; and lane D is 28 day old fibers.
 EXAMPLE 10
 Expression of Transgenic Melanin Synthesis Genes
 A binary construct for plant transformation to express genes for melanin
 synthesis is prepared as follows. The mel operon of Streptomyces
 antibioticus (Bernan et al. (1985) 34:101-110) is subcloned as a BclI
 fragment into a Bluescript vector. NcoI and BamHI sites are inserted by
 mutagenesis immediately S' to (and including) the ATG initiation codon for
 0RF438. The resulting plasmid is pCGN4229. pCGN4229 is further mutagenized
 by inserting a PstI site immediately following the 0RF438 stop codon and
 by the addition of NcoI and BamHI sites at the start codon of the AroA
 locus, thus, providing the mutagenized mel operon. A PstI site from the
 plasmid vector is similarly located immediately 3' to the tyrA encoding
 region.
 The pZ130 cassette, pCGN2909, is mutagenized to reinsert the NcoI site
 including the ATG codon for the initial MET of the pZ130 encoded sequence,
 and results in pCGN4228. pCGN4228 is mutagenized to delete the BamHI site
 at the 3' end of the pZ130 transcriptional termination region and to
 insert an AscI linker fragment in its place, resulting in pCGN4235. Other
 plasmids were mutagenized to delete the BamHI site but had no AscI linker
 substituted in its place. These were designated pCGN4236. pCGN4236 was
 then mutagenized to insert an AscI linker 5' to the pZ130 transcriptional
 initiation region (at XhoI/SalI digested and Klenow treated) resulting in
 pCGN4241.
 The Streptomyces 0RF438 region is obtained by digestion of the mutagenized
 mel operon construct with NcoI and PstI and inserted into NcoI/PstI
 digested pCGN4235. The tyrA region is cloned as an NcoI/PstI fragment from
 the mutagenized mel operon construct into Nco/Pst digested pCGN4241.
 A fragment of the tobacco ribulose bisphosphate carboxylase small subunit
 gene encoding the transit peptide and 12 amino acids of the mature protein
 is inserted in reading frame with the ORF438 encoding sequence as an
 NcoI/BamHI fragment. The fragment is similarly inserted in front of the
 tyrA encoding sequence. The resulting constructs contain the transit
 peptide/ORF438 and transit peptide/tryA fusions positioned for expression
 from the pZ130 5' and 3' regulatory regions.
 A binary vector (See FIG. 7) for insertion of the ORF438 and tyrA
 constructs is prepared from pCGN1578 (McBride et al., supra) by
 substitution of the pCGN1578 linker region with a linker region containing
 the following restriction digestion sites:
 Asp718/Asc/Pac/XbaI/BamHI/Swa/Sse/HindIII. (See FIG. 8). This results in
 pCGN1578PASS. Asc, Pac, Swa and Sse are restrictive enzymes that cut at
 8-base recognition sites. The enzymes are available from New England
 BioLabs: Asc, Pac; Boehringer Manheim:Swa; and Takara (Japan) :Sse.
 The 0RF438 pZ130 construct is inserted into pCGN1578PASS as an Asp/Asc
 fragment. The tyrA pZ130 construct is inserted adjacent to the ORF438
 pZ130 construct as an Asc/Xba fragment.
 EXAMPLE 11
 Preparation and Analysis of Plants Transformed with pZ130 Expression
 Constructs pCGN2905 and pCGN2925
 Preparation of pCGN2925
 The expression construct (described above in Example 6) containing, in the
 5' to 3' direction of transcription, a 1.8 kb ovary tissue promoter
 derived from Lambda 140, a tmr coding region and tmr 3' transcriptional
 termination region (and designated pCGN2903), was modified as follows to
 create pCGN2925. The 2.8 kb EcoRI fragment from pCGN2903 was inserted into
 the EcoRI site of pCGN2015 creating pCGN2910. The 3.2 kb KpnI to SalI
 fragment of pCGN2059 was then inserted into the KpnI and SalI sites of
 pCGN2910 creating pCGN2922. The 6 kb KpnI to XbaI fragment from pCGN2922
 was then inserted into the KpnI and XbaI sites of the binary cassette
 pCGN1557 to create pCGN2925. pCGN2925 is thus identical to pCGN2905 (the
 designation for the plasmid containing the XbaI to KpnI fragment from
 pCGN2903 inserted into the binary cassette pCGN1557 described in Example 6
 of the current application) except that it includes an additional
 approximately 3.2 kb of pZ130 DNA sequence upstream of the SalI site
 located at position 808 of FIG. 2.
 Preparation of Transgenic Plants
 Transgenic cotton plants of the Coker 130 variety were transformed using
 pCGN2905 and pCGN2925, in the method as described in Example 9. Transgenic
 tomato plants of the inbred breeding line UC82B were transformed using
 pCGN2905 as described in Example 7 of U.S. Pat. No. 5,177,307.
 Analysis of Transgenic Plant Agronomic Traits
 Cotton plants transformed with pCGN2905 and pCGN2925, confirmed through
 expression of the gene conferring resistance to the antibiotic kanamycin,
 have been obtained. A greenhouse experiment conducted with the segregating
 offspring of several original transformants supported the conclusion that
 increasing levels of cytokinin in cotton ovaries increases fruit set/fruit
 retention. The number of anthesis flowers in five non-transformed control
 plants varied between 30 and 53; the two offspring from transformant
 2925-2 had 83 and 96 anthesis flowers. The number of bolls on the controls
 remaining at harvest varied between 20 and 38; the two 2925-2 plants had
 66 and 58 bolls at harvest and offspring from 2905-2 had 46 and from
 2905-3 had 45 bolls at harvest. Transformed tomato plants, confirmed
 through a Southern analysis and homozygous for the pZ130/tmr/tmr chimeric
 gene from pCGN2905, have also been obtained. Examination of fruit weight
 data in combination with plant yield information from a replicated field
 analysis indicated that increased levels of cytokinin in ovaries had
 apparently increased fruit set in at least two of the transgenic lines
 tested. The number of fruit per meter of plants in a plot was considerably
 higher for lines 2905-9 and 2905-18 than for the non-transgenic controls
 (approximately 207 and 192 versus 162, respectively).
 Analysis of Transgenic Boll and Fiber Traits
 Analysis of fiber samples from cotton plants transformed with pCGN2905 and
 pCGN2925 indicated that fiber quality characteristics had been altered as
 compared to fiber from the non-transformed control plants. For example,
 fiber length measurements in the control plant fiber varied between 1.12
 and 1.15 inches; fiber length in plant 2905-2A was 1.17 and in plant
 2905-3B was 1.19. Fiber strength in the control samples varied between
 24.3 and 25.7 grams/tex; fiber strength in plant 2905-2B was 26.8 and in
 plant 2905-3B was 27.6 grams/tex.
 Micronaire measurements in the controls varied between 4.4 and 4.8.
 Micronaire measurements in the three 2905-2 offspring examined were 4.0,
 3.5 and 4.0 and in plant 2905-3B the micronaire measurement was 4.2. (For
 a description of micronaire, see Munro, Cotton, 2d ed.(1987) Longman
 Scientific & Technical, Essex, England).
 Other fiber quality measurements in the two 2925-2 offspring were also
 significantly altered when compared to the measurements made for control
 plants. Lengths of 1.09 and 1.06 and strengths of 20.6 and 19.3 were
 measured in fiber samples from plants 2925-2A and 2925-2B, respectively.
 Analysis of fruit samples from tomato plants transformed with pCGN2905
 indicated that fruit quality characteristics had been altered as compared
 to fruit from non-transformed control plants. For example, levels of total
 fruit solids were significantly increased in six of seven independent
 transgenic tomato lines examined (5.79 in controls versus 6.27-6.72 in the
 six transformants). Levels of soluble fruit solids were significantly
 increased in five of seven independent transgenic tomato lines examined
 (5.18 in controls versus 5.68-5.96 in the five transformants). The sugar
 to acid ratio of fruit samples was also significantly increased in six of
 seven of the independent lines (9.3 in controls versus 10.5-12.0 in the
 six transformants).
 EXAMPLE 12
 Preparation of Expression Cassette Constructs pCGN2937 and pCGN2939
 Preparation of pCGN2931 and pCGN2932
 The plasmid pCGN2931 contains the sequences from position 3,288 through
 5,810 from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955 (as
 sequenced by Barker et al., Plant Mol. Biol. (1983) 2:335-350). This
 region contains the entire coding region for the genetic locus designated
 tms-2 (or iaaH) which encodes indoleacetomide hydrolase (Schroder et al.,
 Eur. J. BioChem. (1983) 138:387-391; Thomashow et al., Pro. Natl. Acad.
 Sci. (1984) 81:5071-5075), 347 bp 5' of the translation initiation ATG
 codon and 772 bp of sequences 3' to the translation stop TAG codon.
 pCGN2931 was created as follows. DNA from pTi15955, or any other plasmid
 containing the iaaH locus from the T-DNA region of pTi15955, was used as
 template in a standard polymerase chain reaction (PCR) using
 5'-CGCGGGTCGACTGCAGTGTTAGAAAAGATTCG-3' (SEQ ID no:7) and
 5'-CGGACTCTAGAGATGTGAGGTGTG-3' (SEQ ID no:8) as primers.
 The resulting approximately 2.5 kb fragment was isolated, cut with
 restriction enzymes SalI and XbaI and inserted into the SalI and XbaI
 sites of plasmid pBluescript KS II- (Stratagene). The resulting plasmid is
 pCGN2931. The 2.5 kb PstI fragment from pCGN2931 was then inserted into
 binary cassette pCGN1557 creating pCGN2932.
 Preparation of pCGN2930
 The plasmid pCGN2930 contains the sequences from position 5,809 through
 8,076 from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955 (as
 sequenced by Barker et al., Plant Mol. Biol. (1983) 2:335-350). This
 region contains the entire coding region for the genetic locus designated
 tms-1 (or iaaM) which encodes tryptophan monooxygenase (Thomashow et al.,
 Science (1986) 231:616-618). pCGN2930 was created as follows. DNA from
 pTi15955, or any other plasmid containing the iaaM locus from the T-DNA
 region of pTi15955, was used as template in a standard polymerase chain
 reaction (PCR) using 5'-CGAATCTGCAGATGTCAGCTTCACC-3' (SEQ ID No: 9) and
 5'-CGGGGCTGCAGCTAATTTCTAGTGC-3' (SEQ ID No: 10) as primers. The resulting
 approximately 2.3 kb fragment was isolated, cut with restriction enzyme
 PstI and inserted into the PstI site of plasmid pBluescript KS II-
 (Stratagene). The resulting plasmid is pCGN2930.
 Preparation of pCGN2934 and pCGN2936
 The plasmids pCGN2934 and pCGN2936 contain 5 kb and 1.8 kb, respectively,
 of DNA 5' of the translational start site of the pZ130 gene, the
 approximately 2.3 kb coding region of iaaM, and the 3' region (from the
 TAA stop codon to a site 1.2 kb downstream) of the pZ130 gene. These
 plasmids were created as follows.
 The 2.3 kb PstI fragment from pCGN2930 was inserted into the PstI site of
 pCGN2928. The resulting plasmid, having iaaM in the sense orientation with
 respect to the pZ130 gene promoter region, was named pCGN2934.
 The 2.3 kb PstI fragment from pCGN2930 was inserted into the PstI site of
 pBCKSII- (Stratagene) creating pCGN2935. The 2.3 kb PstI fragment of
 pCGN2935 was then inserted into the PstI site of pCGN2909. The resulting
 plasmid, having iaaM in the sense orientation with respect to the pZ130
 gene promoter region, was named pCGN2936.
 Preparation of pCGN2937 and pCGN2939
 The pZ130 /iaaM/pZ130 constructions were then excised from pCGN2934 and
 pCGN2936 as XbaI to KpnI fragments (of 11 kb and 7.8 kb, respectively) and
 cloned into the binary vector plasmid pCGN2932 (already containing iaaH).
 The resulting plasmids are designated pCGN2937 (1.8 kb pZ130 promoter) and
 pCGN2939 (5.0 kb pZ130 promoter).
 As shown by the above results, expression of a gene of interest can be
 obtained in cells derived from ovary cells, including tomato fruit and
 cotton fibers.
 All publications and patent applications cited in this specification are
 herein incorporated by reference as if each individual publication or
 patent application were specifically and individually indicated to be
 incorporated by reference.
 Although the foregoing invention has been described in some detail, by way
 of illustration and example for purposes of clarity and understanding, it
 will be readily apparent to those of ordinary skill in the art that
 certain changes and modifications may be made thereto, without departing
 from the spirit or scope of the appended claims.
 SEQUENCE LISTING
 (1) GENERAL INFORMATION:
 (iii) NUMBER OF SEQUENCES: 10
 (2) INFORMATION FOR SEQ ID NO: 1:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 564 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA to mRNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1
 AAA AAA ACA AAA ACA TTT CTA ATC TTT TTC ACT CAT TCC ATG GCT CGT 48
 Lys Lys Thr Lys Thr Phe Leu Ile Phe Phe Thr His Ser Met Ala Arg
 1 5 10 15
 TCC ATT TTC TTC ATG GCA TTT TTG GTC TTG GCA ATG ATG CTC TTT GTT 96
 Ser Ile Phe Phe Met Ala Phe Leu Val Leu Ala Met Met Leu Phe Val
 20 25 30
 ACC TAT GAG GTA GAA GCT CAG CAA ATT TGC AAA GCA CCA AGC CAA ACT 144
 Thr Tyr Glu Val Glu Ala Gln Gln Ile Cys Lys Ala Pro Ser Gln Thr
 35 40 45
 TTC CCA GGA TTA TGT TTT ATG GAC TCA TCA TGT AGA AAA TAT TGT ATC 192
 Phe Pro Gly Leu Cys Phe Met Asp Ser Ser Cys Arg Lys Tyr Cys Ile
 50 55 60
 AAA GAG AAA TTT ACT GGT GGA CAT TGT AGC AAA CTC CAA AGG AAG TGT 240
 Lys Glu Lys Phe Thr Gly Gly His Cys Ser Lys Leu Gln Arg Lys Cys
 65 70 75 80
 CTA TGC ACT AAG CCA TGT GTA TTT GAC AAA ATC TCA AGT GAA GTT AAA 288
 Leu Cys Thr Lys Pro Cys Val Phe Asp Lys Ile Ser Ser Glu Val Lys
 85 90 95
 GCA ACT TTG GGT GAG GAA GCA AAA ACT CTA AGT GAA GTT GTG CTT GAA 336
 Ala Thr Leu Gly Glu Glu Ala Lys Thr Leu Ser Glu Val Val Leu Glu
 100 105 110
 GAA GAG ATT ATG ATG GAG TAA TAA TTA AGT GAG GTT AAA TAA GGA TTT 384
 Glu Glu Ile Met Met Glu Leu Ser Glu Val Lys Gly Phe
 115 120 125
 TGA GTG TCA AAA AAA ACA AAA TTA ATA AAG TGT TGC CTT TTC TTA TTA 432
 Val Ser Lys Lys Thr Lys Leu Ile Lys Cys Cys Leu Phe Leu Leu
 130 135 140
 GGG TAG CTT GTG ATG TTG TGT TAG TAT TGG CCT ATA GTA GCC ATT TGA 480
 Gly Leu Val Met Leu Cys Tyr Trp Pro Ile Val Ala Ile
 145 150
 CAC ATT AAA TAA GTT TGT GAC ACA TCA TTA ATC CTT ATG TAT GTA TGT 528
 His Ile Lys Val Cys Asp Thr Ser Leu Ile Leu Met Tyr Val Cys
 155 160 165
 TTT AAT GAA AAA TGA TCG ACT ACG ATC TTT AAT TTT 564
 Phe Asn Glu Lys Ser Thr Thr Ile Phe Asn Phe
 170 175
 (2) INFORMATION FOR SEQ ID NO: 2:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 4383 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA to mRNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2
 GCTCCACTAC TCTCATCACT TTAGTTCATC AAGCCTTCTT TTATACCAAG GCATCATCAA 60
 TCTCATTAAC AAAGTAGATT AGGGTTTTTC AAGATTTAGG ATTCAATAGC TTCATCATGC 120
 TTATTTTATC ACAATTATAT AATCACATTC ATACAAGCAT ACAATTAAGC ATATAGAAGG 180
 GTTTACAATA CTACCCAATA CATATCATTC GCTATTAAGA GTTTACTACG AATAGCATAA 240
 ACCATAACCT ACCTCCACCG AAGAATCGCG ATCAAACAAT CTACTTTCCC AAAGCTGCGT 300
 TCTTCTTCGT TTTCTCTCTC TCTTGATCGT TCGTTTCTCC CTCTCTTTGT TCTTTCTATT 360
 TTTCTTATTC AAACCCTCTT TCTTTTACCC TAATTAGTAT ATAATTAAGT ATAAAAGATG 420
 ATAAAATACC CCATCTATTT GTTTGAAGGT TATCTCTTTT AGCCCCCAAG TAATTGAATT 480
 ATTAACATTA AACCACTAAC TTTATAATTA TAAGCAGGAA TAGTCCAAAA CGCCCCTTAA 540
 AATATTTAAC AGAAATCCGA CCCAGTCAGG GTCACGCAGC CTGTANCGGN NCACAACTGT 600
 GACGGTCCGT CCTGCATGGC CGTCACAAAG TTCAGAGAGT TAATTTCTGT GGAAGATGTG 660
 TANGGTNGTC GTGCCCACGA CGGTCCGTCC TGTCATTTCG TTACGAAGTT CAGAGAGTCG 720
 ATTTCAGTAC CCAAATTTCA GAATTCTAAG TGTTTTGGAA CGAGACCCCN CGGTCCGTCG 780
 TGCCCATGAC GGTTCGTCGT GGGATCCGTC GACTCAGCCA GTTTTTCCAA AATTAAAATC 840
 TGCTGCTCAA AACGACTAAA CAGGTCGTTA CAAAGTACTC AATCAAATAA AAAGAATAAA 900
 TTCTTTTCCA AATACATATA TTGTTTATAG GACAGTGTTA ACAGGGAAAT GTAATCGTTG 960
 CCTCAATCGA TTTTTTTTTT TGAAATTAAG ATTGATTAGA TCTTCTTTAA GATAACAATG 1020
 TCTCAAAGAT AAATTGAATG AATGAATTAG CTATATTATC ATTTGAAAAG AAATTACTAA 1080
 AACAGATTGA TAATAAAATA ATAATAAATG ACTTTGCATC TAAAATAGCT AGAAAGCAGA 1140
 TTTTTAAATA AAAATACATA TGATAAAAAA AAGATAAATT AGAGTCATCC CATAAATTTC 1200
 GCTTTAGGCC CCCAATGTTG TTAAGTCGGC CCTGAAAATA GGAATGGTAT TAAATATTTT 1260
 GTTTTGATTT CACACTTGAT ATTTGACATT CATATTAGAA AATAATTAAA TTTATATTCG 1320
 TGTAGAGTGG TCTCACATTA ATGGGTAAAA TATTTCCACA CAAAAACTAT TTTACAATCA 1380
 TAGCTAGAAT CTGAAATATC TAATGTACTC CACCCAATTA ATTAAAGATG ATTTTTTTGC 1440
 TTAAATAATA AAAATATGTC TATTGCCAAA CTACTAATAG ATGTACTCAC AAAAAAAATA 1500
 AAATAAAAAA TCAAGTGTAT ATACAATGAT TCGGAAGGCC ATTTTTGAAA ATTTTCATAA 1560
 AATGACCGTT TTACCCGTTC ACAATTGTTG TTTCAGCATT TTTGTTTGGT TTGTGGATTT 1620
 GGTTATGGAA GTTCAATAAA AAGTTGTGGT TTTATAAGCT TTGGAGTTTT GAAAGGTTTA 1680
 AGTTGATTAA AAGTAGTTTT TAGTGTCAAT TGGAGTTTCG TGTCTTGAAA TAAATTTTAT 1740
 CACTTGCATT AGTTTCAAAA TGTCGAGTTT GGTTAAGTAG AGGTTTTTTT CATTCGGAGT 1800
 TTTTTTATGA ATTTAAAATG TTAAGCTGAA AGTTTATGAA ATTTTAGCCT TTGAGTTAAT 1860
 TTTGATGCTT GAATTAAATT TTTGAGAATT TTTTTGAAAT CTGGGGATAA TGTTAGGTCT 1920
 TAGAGAAGTC TGGTTGAATT TTCATAGCTC AAGAGATTAG TTTTGACTTT TTAGGCATTT 1980
 TGTTGGTTTA TTACGATTTT CACGGACTTT CGAATTAAGG AGACTTCAAA ATTCATATTT 2040
 AATGGTTCGT GTGTTCGTTA GTTTTAAAAA TCGTGTCTTT ATAAGGATTT ATACTTAAAA 2100
 AAATAAAATA AAATAAAGTA CTACTAACAT GTAATTCTGT CATAAGATAA GGTTGTACAT 2160
 TTAGGACTAT TTGAATATTC ATCAAAAATA AAAAAAAGTA GAGATGATAG TAATATAAAT 2220
 ATTTATTTTT GATTTTACAT TTGATATTTT AATACTAACA ATATGACATA ATAAAATTTG 2280
 TATTCAGATT GTAAAATATT CCCTAAAAAA AGATACTTTT ACTGTGGTGG CTCAAATTCA 2340
 AAATTTTCTA AGAAAAACTA CTAATAATTG ATTTCTAATT AAAATTTCGA TATATATATA 2400
 TATATATATA TATATATCAT AATATACTTC ACCTACCTCA ATTATTATTA TTTTCTTTTT 2460
 TTTTTACTTC ACATATTTTT GGSCSACCAA TTTTTTTTTT AACTTTTTTG GTCTTACTCT 2520
 TATTTCACTC CCTATAAATA ACTCCCATTG TGTGATATTT TTATTCACAA CTCTAACTTA 2580
 CAATCTTTCT TATTATTAAA AAAAACAAAA ACATTTCTAA TCTTTTTCAC TCATTCCATG 2640
 GCTCGTTCCA TTTTCTTCAT GGCATTTTTG GTCTTGGCAA TGATGCTCTT TGTTACCTAT 2700
 GGTTTGTCTT CATAATTTAT TCCTCTAAAA TCATCGCAAT AAAAAAAAAA TGTAACGAAG 2760
 CAGACATCAG TAAACCGTTT AAATAAACCC TAAAAAAATT GTGAATTGAT ATTACTTGCT 2820
 ATACGTTTAA CAACTATGAT AAAAAAACCC TAAAATATAC TTATTTCGAT TTCGTCTCTC 2880
 TCATGTTATT CTAACTATTT TTTGTGTGTG AATGATTGTA GAGGTAGAAG CTCAGCAAAT 2940
 TTGCAAAGCA CCAAGCCAAA CTTTCCCAGG ATTATGTTTT ATGGACTCAT CATGTAGAAA 3000
 ATATTGTATC AAAGAGAAAT TTACTGGTGG ACATTGTAGC AAACTCCAAA GGAAGTGTCT 3060
 ATGCACTAAG CCATGTGTAT TTGACAAAAT CTCAAGTGAA GTTAAAGCAA CTTTGGGTGA 3120
 GGAAGCAAAA ACTCTAAGTG AAGTTGTGCT TGAAGAAGAG ATTATGATGG AGTAATAATT 3180
 AAGTGAGGTT AAATAAGGAT TTTGAGTGTC AAAAAAAACA AAATTAATAA AGTGTTGCCT 3240
 TTTCTTATTA GGGTAGCTTG TGATGTTGTG TTAGTATTGG CCTATAGTAG CCATTTGACA 3300
 CATTAAATAA GTTTGTGACA CATCATTAAT CCTTATGTAT GTATGTTTTA ATGAAAAATG 3360
 ATCGACTACG ATCTTTAATT TTATGTTTTA CATTTAATTA ATCACTTTCT GTTACGATTC 3420
 ATTTATCTAG TTATGAATGA AATATAGAGT GATTTGAAGT AAGGAGCTAG TCTTCAAACA 3480
 AAGACGTACA TATGTACAAA GTAGGGTACT ATTAAACTTC TTTTTTATGA TTCGATATAT 3540
 TCATATTTGA TACTCAAATT AGAGTTAAAT TCATATTAAT TTGTACGAGA AATTTTAAAC 3600
 TAATAAATAA AACTCTCCCT AATAAAAGAT TACTTTCATG ATAGAATATA TAATATTATG 3660
 AATTCTTGTT GCTGAATTTA TATTATGGTC ATGCAAAACT TAGGAAAATA AAATGAAAGA 3720
 TAAATAATAT GTGCTTGATG ACAACTTTAT GCCTGAATTA ATATAATAAT AATTAATATA 3780
 AAATGATGAA TTAATAATAC TTTATAATAA GTTTTTTCTT CATCATTTGA ATCTATTGAA 3840
 TGGTATTAAA CATTTTATTT TGATTTTACA TTCGATATTT GATATTTATA ATAGAAAATG 3900
 ATTAAATTTA TATTCGTTTA GAGAGGTCTC ATATTAAAGG ATAAAATATT ATCTAATAAA 3960
 AGTTACTTTA CAATCATAGT TAAAATCTGA AATATCTAAT GTTGTAATGA CCCGATAGAT 4020
 TATTTTTGGG AATTTTAAAC TATTTGCTTA ACTTAAGTTA ATTAATTCAT GAATAATTAT 4080
 AGATAATTGA CTTAATCAAT AGTTAATGAT ACAACTATAT ACTATTTGAC CCTTATAATG 4140
 TTGTGTTAAA TATTGGTCTT TAGTAGCCAT TTGACACATT AAATAAGTAT TGCATAAATG 4200
 TTTTACCAAA AAAAATCAAT TAGAAATCAT ATCAGTAAAT ATTCATGAGC TAATACCTAA 4260
 AATAAAATTG AAAAAAAAAT CAAATAATTT CAATTCATCG ATTTTGCAAA AATTATGCAG 4320
 AAAAATTAAA CAATTGCACA ATCCATCAAT TTAGCATTAA GTATTTAGCC CTCTCTTGGA 4380
 TCC 4383
 (2) INFORMATION FOR SEQ ID NO: 3:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 453 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA to mRNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3
 ATTATTATTA CC ATG GCA CAA AAA TTT ACT ATC CTT TTC ACC ATT CTC CTT 51
 Met Ala Gln Lys Phe Thr Ile Leu Phe Thr Ile Leu Leu
 1 5 10
 GTG GTT ATT GCT GCT CAA GAT GTG ATG GCA CAA GAT GCA ACT CTG ACG 99
 Val Val Ile Ala Ala Gln Asp Val Met Ala Gln Asp Ala Thr Leu Thr
 15 20 25
 AAA CTT TTT CAG CAA TAT GAT CCA GTT TGT CAC AAA CCT TGC TCA ACA 147
 Lys Leu Phe Gln Gln Tyr Asp Pro Val Cys His Lys Pro Cys Ser Thr
 30 35 40 45
 CAA GAC GAT TGT TCT GGT GGT ACG TTC TGT CAG GCC TGT TGG AGG TTC 195
 Gln Asp Asp Cys Ser Gly Gly Thr Phe Cys Gln Ala Cys Trp Arg Phe
 50 55 60
 GCG GGG ACA TGT GGG CCC TAT GTT GGG CGC GCC ATG GCC ATA GGC GTG 243
 Ala Gly Thr Cys Gly Pro Tyr Val Gly Arg Ala Met Ala Ile Gly Val
 65 70 75
 TGATTACAAT TTCGTTGTTC TTCTTTTTCG ACTTTTTAAT CCCAAGTGAA TAAAGTCTAA 303
 TTCGAAAAAG AAGAAAAAAG TATCTATGTC TGAGTYATAT GTTTTGTGGC TAATAAGAAA 363
 TCGACTATGC TTGTTGATTT GATAAAAATT ATGTCATTAG GGTGTGATAT GTAATCATCA 423
 AATTAAATAA AAATCATCGC ATTGTGTGTG 453
 (2) INFORMATION FOR SEQ ID NO: 4:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 36 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other
 (A) DESCRIPTION: synthetic oligonucleotide
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4
 AATTAGATGC AGGTCCATAA GTTTTTTCTA GACGCG 36
 (2) INFORMATION FOR SEQ ID NO: 5:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 42 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other
 (A) DESCRIPTION: synthetic oligonucleotide
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5
 GTTCCTGCAG CATGCCCGGG ATCGATAATA ATTAAGTGAG GC 42
 (2) INFORMATION FOR SEQ ID NO: 6:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other
 (A) DESCRIPTION: synthetic oligonucleotide
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6
 CAAGAATTCA TAATATTATA TATAC 25
 (2) INFORMATION FOR SEQ ID NO: 7:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 32 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other
 (A) DESCRIPTION: synthetic oligonucleotide
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7
 CGCGGGTCGA CTGCAGTGTT AGAAAAGATT CG 32
 (2) INFORMATION FOR SEQ ID NO: 8:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 24 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other
 (A) DESCRIPTION: synthetic oligonucleotide
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8
 CGGACTCTAG AGATGTGAGG TGTG 24
 (2) INFORMATION FOR SEQ ID NO: 9:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other
 (A) DESCRIPTION: synthetic oligonucleotide
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9
 CGAATCTGCA GATGTCAGCT TCACC 25
 (2) INFORMATION FOR SEQ ID NO: 10:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other
 (A) DESCRIPTION: synthetic oligonucleotide
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10
 CGGGGCTGCA GCTAATTTCT AGTGC 25