MXPA98009118A - Method and transgenic compositions to produce fruits and vegetables partenocarpi - Google Patents

Abstract

The invention describes a transgenic method for producing parthenocarpic fruits or fruits with a reduced number of seeds, involves the temporary expression of a phytohormone, presureor or a gene, so that the activity of gibberellin or other similar hormonal activity involved in the start of the fruit formation activity, the gene is operably linked to a regulatory promoter, so that the expression is synchronized before the development of pollen or fertilization, the expression of the hormone causes fruit development in the absence of fertilization; The method also results in a fruit that has small seeds or very few seeds, the invention also includes constructions, vectors and transgenic methods for the production of parthenocarpic plants.

Description

METHODS AND COMPOSITIONS TRANSGEN I CQS TO PRODUCE FRUITS AND HOTILTA S? S PtAftTENQC PEGOS GAMPP AND I INVENTION This invention relates generally to the field of plant molecular biology »and in particular to plants» transgenic seeds and tissues »which have been genetically modified to create plants which will avoid the need for fertilization and the initial formation of the fruit and which» without considering these last »will produce fruits and vegetables without seeds under various growing conditions.

NTSCEPENTES PE l »ENVENCtON In angiosperm plants »the fruit develops from the pericarp» that surrounds the developing seed. The development of the fruit occurs after pollination and fertilization "which results in the production of an embryo. After the fusion of the male gamete with other cells in the ovule, the formation of the endosperm begins and develops concomitantly with the proliferation of the protoplast embryo. As the ovules develop in the seeds, the ovary swells in the fruit. Often "the development of the fruit in non-apoptotic species is triggered by the fertilization of the ovules" and if pollination or fertilization does not occur "then abscission of the flower occurs and no fruit is formed. The formation of the fruit is also hampered by external factors such as temperature fluctuations, availability of water and light conditions. For example, peppers are very sensitive to temperature fluctuations. A temperature lower than 10 ° C during the night or higher than 26.6 ° C during the day »will cause the fruit not to form after pollination. Minor variations will lead to the uneven formation of the fruit »resulting in fruits of abnormal shape. The possibility of controlling the start of the formation of the fruit in the plants can avoid these problems "since the formation of the fruit can be controlled internally without importing the external factors" thus improving the yield "as well as the areas of potential growth. Said control can also be provided for the start of the formation of the fruit without fertilization, that is to say the production of an enoharpic pair phenotype. This may allow the development of the fruit by a vegetable species without considering the external factors "and may allow the development of the fruit without considering the fertilization. Frequently, the "parteñocarpico" phenotype not only allows the formation of the fruit without fertilization, but without fertilization. Seed development is minimized or completely inhibited, providing a second benefit of parthenocarpy. Seedless fruits and vegetables have long been an objective of those in the field of agricultural products. The benefits of such plants include the obvious attraction for consumers of the facility to prepare and consume such products. Other benefits include a sweeter and fresher fruit or vegetable "as well as an increase in the edible portion" whenever the seed cavity is absent or greatly reduced. Several advances have been made in this field »usually with topical application of hormones or with highly complex reproductive procedures that result in a triploid genotype. Topical applications of gibberellin have been used for a long time to induce a seedless phenotype < partenocárico) in grapes. This method usually requires a spraying procedure that is problematic and climate dependent and must be correctly synchronized to occur immediately after pollination. Another method requires a complex breeding procedure for the production of seedless watermelons. The seedless condition in watermelon is almost always the result of the presence of three homologous supplements per cell "instead of the two normal ones" known as triploid. These watermelons have problems to develop normally in an embryo and cause the absence of seeds in the plant ipioides. The abnormal formation of the embryo causes the cessation of the normal ovular development of a seed at an early stage. Typically, seedless watermelons contain small edible white eggs similar to those of immature cucumbers. A transgenic method to create seedless fruits is described in the world patent W091 / 09957. It includes an excision system for highly complex recombination called "CRE-LOX". Generally »the genetic product of CRE produces a pratein recobinase that acts on the specific LOX DNA sequence. Several recombination functions are described »the most consistent being the excision of the LOX DNA sequence. The application hypothesizes that seedless watermelons occur under conditions that include transformation with the barnase gene derived from Baci llus amylol iguefaciens. Generally »the female seed has a specific tegument promoter (SC)? CRE * terminator, and the male seed has a SC? gene barnase promoter? ? what? ? in Barnase? finished . There are no phenotypic changes in the two parents when they are propagated "and in the field of seed production the female seed producer parent has a normal set of seeds because both genes are not working at the same time (the tegument is only maternal tissue). However, in the Flr generation the maternal tissue will contain both genes. In this configuration »the constitution of the tegument (plant Fl» seeds F2) is promoter SC? Gene barnase?; -? in Barnase? terminator »where ?? - ?? represents the point where the lox gene is cut off »producing a gene for functional toxin specific to the tegument. In theory, if the tegument collapses, the development of the seed will be stopped due to the lack of nutrient flow to the embryo. No experimental data are described »and the success of the proposed protocol for the development of real seedless watermelons is nowhere to be found. In this way »it can be observed from the above» that there is a need in the art for production protocols for parthenocarpic fruits and vegetables »that are simple» direct and easily repeatable. An object of the present invention is to provide expression constructs »that when expressed in a transgenic plant» result in the formation of fruits without fertilization »as well as the production of fruit and vegetables without seed. It is also another object of the invention to provide lines endagámicas progenituras that can be reproduced in cross form "giving co or result a plant Fl that will be part arpex and will not produce seeds or will exhibit a significant reduction in the amount of seeds present. It is also an object of this invention to provide plants »plant cells and plant tissues containing the expression constructs of the invention. A further object of the invention is to provide vehicles for the transformation of plant cells "including viral vectors or plasmids and expression cassettes that incorporate the genes and promoters of the invention. It is also another object of the invention to provide bacterial cells comprising said vectors for plant maintenance and transformation replication. Other objects of the invention will become apparent from the following description of the invention.

BRIEF DESCRIPTION OF THE INVENTION In one embodiment, the invention comprises the genetic manipulation of plants to enhance the effects of gibberellins or other hormones involved in the initiation of fruit formation. The invention comprises the temporal expression of a structural gene encoding a phytohormone such as gibberellins or cytokines or proteins associated with the production of said hormones (ie »enzymes» biosynthetic intermediates »etc.» which are associated with the initiation of the formation of the fruit. The structural gene is put under the control of a specific promoter of the icrospore or megaspore of the pollen "so that the expression of the hormone is synchronized so that it occurs before pollination" so that the development and maturation of the fruit are induced without the need for fertilization.

PE5GRIPGIQN PETAi Pft PE * I VENTION The topical use of exogenous hormones to facilitate the formation of the fruit "or in some cases to induce the tenocarpia" is well established in the areas of fruit and vegetable cultivation. The use of the auxin inhibitor Tomaset (N-meta-toli-l-phthalamic acid or 4-CPA) had previously been achieved to induce parthenocarpy in tomatoes under cold conditions in greenhouses. Pepper growers in Florida use the GA application to improve fruit formation during the growing season. The vines are usually treated with gibberellins to induce the formation of the fruit »lengthen the size of the clusters and uniform the growth of the fruit» especially in the case of the seedless types. See »for example» Jankiewicz »L.S.» Flowers »A.E.r Gsrec i» R. »Staniaszek» M »" Effect of growth regulators on parthenocarpic fruit in Capsicum annum ". The additional treatment with gibberellin is used to ensure that the new fruit is formed as the previous fruits mature and are harvested. See »SO? Folia-Hor ticul urae (Poland) (1991) »v. 3 (2) »p. 3-16. In the following description »several terms are used exhaustively. The following definitions are provided to eliminate ambiguities in the intent or scope of their use in the specification and claims and to facilitate understanding of the invention. s As used herein, the term "gibberellin" should include any product that possesses the biological activity of a gibberellin, and may include gibberellin-like proteins having homology to a gibberellin product so that their biological activity is conserved by studies of mutation described later. As used herein, the term "fruit" should include any angiosper that has its pollen and ovule-producing organs in flowers "with ovules enclosed in an ovary" and after fertilization "with each ovule developing into a seed" while the ovary expands in a fruit. Any fruit that is convenient to be produced in a partenactric way "is encompassed by this definition. In addition, it is intended that food sources traditionally considered as vegetables "but which are produced in this manner" such as tomatoes, peppers and the like be encompassed. A promoter is a DNA sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5"region of a gene" near the start site of the transcription of a structural gene.A pollen specific promoter is any promoter capable of regulating temporal expression at a time prior to pollination. or shortly thereafter "so that the development and maturation of the fruit are induced" without significant development of the seed, such promoters as those mentioned here include but are not limited to »inducible promoters» promoters of microspores or megaspores » pollen specific promoters or promoters of maternal tissue »such as tegument promoters» or any other associated promoter with a gene involved in the pollination or maturation or development of the ovule. A structural gene is a DNA sequence that is transcribed into messenger RNA (mRNA) "which is then translated into an amino acid sequence characteristic of a specific polypeptide. The term "expression" refers to the biosynthesis of a genetic product. In the case of a structural gene »the expression involves the transcription of the structural gene into mRNA» and then the translation of the mRNA into one or more polypeptides. A cloning vector is a DNA molecule such as a plasmid »costed or bacterial phage that has the ability to replicate autonomously in a host cell. The cloning vectors typically contain one or a small number of restriction endonuclease recognition sites in which DNA sequences introduced in deter-geable form can be inserted without loss of the essential biological function of the vector "as well as a marker gene that it is suitable for use in the identification and selection of cells transformed with the cloning vector. An expression vector is a DNA molecule that comprises a gene that is expressed in a host cell.

Typically, gene expression is placed under the control of certain regulatory elements that include promoters, tissue-specific regulatory elements, and enhancers. It is said that said gene is "operably linked to" the regulatory elements. A recombinant host can be any prakaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes the prakaryotic or eukaryotic cells that have been manipulated by genetic engineering techniques to contain the cloned genes in the chromosome or genome of the host cell. A transgenic plant is a plant that has one or more plant cells that contain an expression vector. Plant tissue includes differentiated or undifferentiated tissues or plants including »but not limited to» roots »stems» v stems »leaves» pollen »seeds» tumor tissue »and various forms of cells and cultures such as individual cells» protoplasts »embryos and tissue callous. The plant tissue can be in a plant or in a culture of organs, tissues or cells.

MOLECULAR TRANSFORMATION TECHNIQUES The production of a genetically modified plant tissue expressing a structural gene under the control of regulatory promoters "combines the teachings of the present disclosure with a variety of techniques and expedients known in the art. In most cases »there are alternate dossiers for each stage of the overall procedure. The choice of files depends on variables such as the plasmid vector system chosen for the cloning and introduction of the recombinant DNA molecule »the plant species that will be modified» the structural gene in particular »elements of the promoter and elements towards the end 5 'used. Those skilled in the art are capable of selecting and using suitable alternatives to achieve functionality. Culturing conditions for expressing desired structural and cultured cell genes are known in the art. Also "as is known in the art" a number of monocotyledonous and dicotyledonous plant species are transformable and regenerable so that whole plants can be obtained which contain and express suitable genes under the regulatory control of the promoter molecules according to the invention. As is known to those skilled in the art »expression in transformed plants can be tissue specific and / or specific for certain stages of development. Truncated promoter selection "and selection of the gene for structural hormone" are other parameters that can be optimized to achieve the desired plant expression "as is known to those skilled in the art and taught herein.

The selection of a suitable expression vector will depend on the method for introducing the expression vector into host cells. Typically, an expression vector contains (1) prokaryotic DNA elements that code for a bacterial origin of replication and a marker of antibiotic resistance to provide growth and selection of the expression vector in a bacterial host »(2) elements of DNA that controls the initiation of transcription "such as a promoter" (3) DNA elements that control the processing of transcripts »such as the sequence of termination / polyadenylation of transcription» and (4) a reporter gene that is linked operably to the DNA elements that control the start of transcription. Useful reporter genes include β-glucuronidase »0-galactosidase» chloramphenicol acetyl transferase »luciferase» and the like. Preferably, the reporter gene is fl-glucuronidase (GUS) or luciferase. General descriptions of expression vectors and reporter genes in plants can be found in Gruber et al. "" Vertors for Plant Transformation "in Methods in Plant Molecular Biology et Biotechnology" "in Glich et al. (Eds. 89-119 »CRC Press» 1993). Furthermore, the expression vectors for GUS and the gene cassettes for GUS »are available from Clone Tech Laboratories» Inc. »Palo Alto» California »while the expression vectors for luciferase and the gene cassettes for the same are available from Pro Mega Corp. (Madison »Wisconsin). Expression vectors containing genomic or synthetic fragments can be introduced into the protoplast or into intact tissues or isolated cells. Preferably, the expression vectors are introduced into intact tissue. General methods for cultivating plant tissues are provided »for example» by Maki and others »" Pracedures for Introducing Foreign DNA into Plants "» in Methods in Plant Molecular Biology &; Biotechnology »Glich et al. (Eds. Pp. 67-88 CRC Press» 1993); and by Phillips and others »" Cell-Tissue Culture and In ~ Vitra Manipulat ion "» on Carn & Corn I provement »3a. edition »Sprague et al. (Eds. pp. 345-387)» American Society of Agrono and Inc. and others »1988. Methods for introducing expression vectors in plant tissues include direct infection or co-culture of plant cells with A robacteriu tumefaciens »Horsch et al.» Science »227: 1229 (1985). Descriptions of the Agrobacterium vector system and methods for Agrobacterium-mediated gene transfer are provided by Gruber et al. Cited above. Preferably, the expression vectors are introduced into the tissues of the plant using a direct method of gene transfer, such as icroprojectile-mediated release, DNA injection, electroporation, and the like. More preferably, the expression vectors are introduced into plant tissues using the release mediated by icroprojectiles with the biolistic device. The invention also comprises the use of these types of transformation procedures to generate a plant in which the formation of the fruit is induced before fertilization "resulting in a parthenocarpic plant that will also contain little or no seed. This is of particular value for plants in which the formation of the seed is inhibited early by external factors such as temperature fluctuations »precipitation» etc. A construction comprising a hormone that stimulates the formation and development of the fruit »or that comprises a gene that codes for a protein that promotes the production of said hormone (a precursor molecule or enzyme) operably linked to a specific pollen promoter "is introduced into said plant. In a preferred embodiment the invention comprises the use of a structural gene which promotes the synthesis of or codes for any of the family of gibberellins or phytohormones similar to gibberellin. The methods of the invention described herein may be applicable to any species of angiasperm whose fruit or vegetable is suitable to be produced in a wide range of conditions without there being any negative impact on the formation and development of the fruit and in which convenient to get the condition without seed. Fruit and vegetable producing plants that can be made seedless in accordance with the methods of the invention include but are not limited to melons such as watermelon and common melon, berries such as strawberries and cranberries, peppers such as green peppers, peppers. red »yellow peppers» tomatoes »oranges» plums »alfalfa» pumpkin »eggplant» sweet corn »peas» cotton »avocados» mangoes »papayas» nectarines »apples» grapefruit »lemons» limes »mandarins» pears and peaches. In a preferred embodiment, the invention is used with fruits such as pepper, in which the formation of the fruit is very sensitive to external conditions. The methods of the invention in relation to particular modalities will be illustrated below. A phytohorn that is critical for the formation and development of the fruit "is one that includes hormones that are critical for the formation and / or function of pollen» icrospores »macrospores or embryos» and includes proteins or enzymes that are fundamental in the development of the fruit »including cells and / or tissues from which the fruit develops» cells and / or tissues that form part of the ovary »endosperm» pericarp or embryo »or other female structure in which the fruit develops after fertilization. The DNA sequence can be any identifiable DNA sequence that codes for genetic products that are capable of inducing the formation and development of the fruit in a cell or a plant. Examples of said DNA sequence include hormones such as gibberellin »auxins» etc. »precursors of said hormones »or enzymes that are involved in the biosynthesis of said hormones. Gibberellins (GAs) are a family of growth hormones terpenoids, some of which are bioactive growth regulators. GAs are required to control processes as diverse as seed germination, elongation and cell division, foliar expansion, stem elongation, flowering and fruit formation. GAs have been the subject of many physiological and biochemical studies »and several mutants of plants with altered biosynthesis patterns have been studied in response to GA (Graebe »J.E.» Ann. Rev. Plant Physiol. 38? 419-465 (1987)). Extensive biochemical studies conducted on endogenous GA intermediates in dwarf mutants that respond to GA »have allowed the determination of the biosynthetic pathway of GA and several genetic loci involved in its biosynthesis (reviewed by Graebe» JE »Ann Rev. Plant Physiol 38 * 419-465 (1987) Several Dwarf mutants have been isolated that respond to GA from several plant species »such as corn» pea and Arabidopsis (Phinney »BO and others» "Chemical Genetics and the Gibberellin Pathway "in Zea mays L." in Plant Grawth Substance »ed.» PF Waering »New York? Academic (1982) pp. 101-110? Ingram, TJ and others» Floor 160? 455-463 (1984) » Koornneef »M.» Arabidopsis Inf. Serv. 15? 17-20 (1978).) Dwarf mutants of maize (dwarf 1 »dwarf 2» dwarf 3 »dwarf 5) have been used to characterize the biosynthetic pathway of GA corn determining specific steps that lead to biologically important metabolites (Ph inney »B.O. and others» "Chemical Genetics and the Gibberellin Pathway" in Zea mays L. »in Plant Growth Substance» ed. »P.F. Waering »New York * Academic (1982) pp. 101-110; Fujiaka »S. and others» Plant Physiol. 88? 1367-1372 (1988)). Similar studies have been carried out with dwarf pea (Pisum sativum L.) mutants (Ingram »T.J. and others» Plant 160? 455-463 (1984)). Mutants deficient in Arabidopsis GA (gal »ga2» ga3 »ga4» ga5) (Oornneef »M. and others» Theor, Appl. Genet, 58? 257-263 (1980)) have also been isolated. One of the most comprehensive genetic studies of GA mutants has been carried out by Koornneef (Theor. Appl. Genet .58? 257-263 (1980)? Koornneef and others »Genet. Res. Ca b. 41? 57-68 (1983)) in the crucifera Arabidopsis thaliana. Using ilomethane sulfonate (EMS) and rapid mutagenesis with neutrons »Koornneef has mapped 9 isolated alleles for the GA1 locus of A. thaliana (Koornneef and others» (Theor. Appl. Genet. 58? 257-263 ( 1980) "Koorneef et al." Genet, Res. Camb. 41? 57-68 (1983).) Structural genes useful for the method of the invention "include genes that encode gibberellin or an intermediary of the biosynthesis thereof" such as an enzyme or precursor that is finally converted into gibberellin In yet another embodiment, the gibberellin receptor can be mutated to make it more sensitive to gibberell.An example of a gene useful in the practice of the invention includes that described in European Patent Application EPO 692537A2 by Tai-Ping Sun and others "Recombinant Gibberellin DNA and Uses Thereof", the disclosure of which is incorporated herein by reference.The publication describes the cDNA and genomic DNA corresponding to the locus of the invention. G The Arabidopsis thaliana coding for ent-kaurene synthetase. The enzyme encoded by the GAl gene is involved in the conversion of GGPP into ent-kaurene (Barendse and Koornneef »Arabidopsis Inf. Serv. 19? 25-28 (1982)? Barendse et al.» Physiol. Plant. 67: 315-319 ( 1986); Zeenvaart »JAD» in Plant Research '86 »Annual Report of the MSU-DOE Plant Research Laboratory» 130-131 (East Lansing »MI» 1986)) »a key intermediary in the biosynthesis of GAs (Graebe» JE » Ann »Rev. Plant Physiol. 38? 419-465 (1987)). Ent-kaurene synthetase has only been partially purified from several plants (Duncan »Plant Physiol., 68? 1128-1134 (1981)). The synthesis of GGPP from mevalonate is common in terpenas. GPPP is a branching point metabalite that is not only the precursor of GAs "but also a precursor to other diterpenes" such as the phytol chain of chlorophylls "and tetraterpenes such as carotenaids. The first obligate step of the GA pathway is the conversion of GGPP into ent-kaurene in a two-step cyclization reaction.

GGPP is partially cyclized to the intermediary »copalyl pyrophosphate (CPP)» by ent-kaurene synthetase A »and the CPP is immediately converted to ent-kaurene by ent-kaurene synthetase B. Since ent-kaurene is a key intermediary in the GA pathway »its synthesis is likely to be a regulatory point for GA biosynthesis. Furthermore, ent-kaurene production has been shown to be altered by changes in the photoperiod »temperature» and the growth potential of tissues in certain species (Chung and Coolbaugh »1986» Moore and Moore »1991; Zeevaart and Gage »1993). Examining the molecular lesions in several GAl alleles, a direct correlation of the genetic and physical maps of the GAl locus was established and a recombination rate of 10 ~ 5 cM per nucleotide was determined for this region of the A. thaliana genome ( Koornneef »Genet, Res. Comb. 41: 57-68 (1983)). Another example of a gene useful for the practice of the invention includes that described in PCT publication W094 / 28141 by Theodor Lange and others "Regulation of Plant Growth", the description of which is incorporated in the present reference. This application describes the molecular cloning of the gene that codes for gibberellin (GA) 20-oxidase and its use. (GA) 20 oxidase is a regulatory enzyme »and the production of GA is particularly sensitive to its activity. The expression of the enzyme increases the levels of biologically active gibberellins. Still other structural genes useful for the invention include the AN1 to ANZ gene described in W095 35383"Plant anther ear genes encoding cyclase affect gibberellic acid biosynthesis-useful for altering height and fertility in monocotyledons" »by Benson» R.J. and others "whose description is incorporated herein by reference. The publication describes the cloning and expression of genes for corn and anther 1 (AN1) and corn and anther 2 (ANZ) cloned from maize. The genetic product affects the conversion of GGPP into ent-kaurene in the biosynthesis of gibberellic acid. A tomato mutant deficient in GA is abnormal »and the result is deformed fruits. This phenotype can be overcome by the application of biactive GAs. Tomato mutations include gibl »gib2» gib3 »none of which has been cloned. The gibl mutation of tomato is the metabolic equivalent of AN1 in corn. The observation that the addition of biaactive GA to develop fruits of gibl »gib2 and gib3 suggests that the expression of a structural gene causes GA to bioactively result in abnormal formation and shape of the fruit in pepper and other fruits. Similar observations have been made in Arabidopsis. In Arabidopsis »the GAl gene is the metabolic equivalent of AN1 in corn» and has been cloned and its sequence determined. In peas »the removal of the seeds at an early stage leads to the abnormal development of the pod» since the source of the bioactive GA is the seed. Thus »in a preferred embodiment» the invention includes the AN1 or AN2 genes which have activity similar to gibberellin to achieve the parthenocarpic phenotype. The loss of AN1 reduces the amount of the metabolic product by 807. of the total produced. The contribution of ANZ in the remaining 207. has not yet been determined. AN2 is a gene expressed with high homology with AN1 (707. identical). Both genes are described in W095 / 35383 »whose description has been previously incorporated as a reference. There is approximately 50 to 60% identity at the amino acid level between AN1 and GAl genes. The sequence of AN1 has been used to recover similar sequences in rice. A comparison of the maize and Arabidopsis sequences can be used to derive a consensus sequence that would be useful for isolating similar genes in pepper or other species for the practice of the invention. Another potential gene includes the GA4 gene of Arabidopsis. These gene products catalyze the last step in the biosynthetic process of gibberellin that makes the GAl bioactive. The gibberellic acids are synthesized from the isoprenaid GGPP »starting with the GGPP sequences in CPP »and then CPP in ent-kaureno» catalyzed by the kaurene synthetases A and B »respectively (Duncan et al. 1981). It is thought that most higher plants are similar to corn because "in corn" ent-kaurene is gradually oxidized to 7-hydroxy-kaurenoic acid "which is converted into the first true gibberellin" aldehyde GA12 (Suzuki and others »1992). The latter compound is then oxidized to an active GA by one of three parallel pathways. In corn, the dominant route seems to be the early 13-hydroxylane route (Hedden et al. 1982), with GAl being the penultimate active product typically present in amounts less than 1 ug / 100 gfwt (Fujioka et al. 1988). The homology between the predicted amino acid sequences of maize AN1 and Arabidopsis GA1 points to a common function for these genes. Its total identity of 47% (68% similarity) is remarkable "but it is even greater in an internal segment of 300 amino acids that is 68% identical (94% similar). As in the case of the putative binding domain of poly prenyl-pyrophosphate within this segment »AN1 and GAl share 100% similarity. Other plant genes whose sequence has been determined and which use poly prenyl-pyrophosphorus substrates (geranyl-phar- nosyl- and geranyl sonyl-pyrophosphate) »also share significant homology with AN1 in this domain (Facchini et al., 1992) »But much less general homology with AN1 (from 20 to 25% identity). These sequence homologies clearly indicate that AN1 codes for a cyclase that functions in the conversion of GGPP to ent-kaurene. The promoters used in the methods of the invention can be a pollen-specific promoter, a maternal tissue promoter, an inducible promoter or a constitutive promoter. It will be appreciated that the term pollen as used herein and in particular in relation to the inducible promoter described in the description and claims includes cells and / or tissues from which pollen develops (eg, pre-icraspores). eióticas and uninucleadas), cells and / or tissues that are part of the masculine structure in which the pollen develops (for example »anther» tapeto or filament) »and the pollen itself. It also includes any promoter that correlates the expression with the early start of the reproduction of the plant as the plant moves towards the vegetative state. This may include promoters associated with fertilization "or the fertile plant including all sexual organs" as well as promoters of the early development of the seed. The specific pollen promoter used can be selected from the group of promoters known to direct expression in the anther »tapeto» filament »pollen itself» a in the ovary »pericarp of the endosper or corresponding embryo structures. Examples of pollen specific promoters include those described in the U.S.A. No. 5 »086» 169 by Mascarenhas »which describes a pollen-specific promoter isolated from corn» the patent of E.U.A. No. 5 »412» 085 for Alien and others »which describes another pollen-specific promoter isolated from corn? the patent of E.U.A. No. 5 »477» 002 by Tuttle and others »which describes a specific promoter of the anther» the patent of E.U.A. No. 5 »470» 359 by Huffman and others »which describes a specific promoter of the tapeto; W092 11379 by Draper et al. »Describing a specific promoter of the isolated Brassicaceae (Braeicaceae) tapeto» Plant »July 1995 8 (1) p. 55-63 »where a specific tobacco pollen promoter is described» and Plant Mol. Biol. »January 1992 18 (2) p. 211-8 »where another specific promoter of corn pollen is still described. These are only some examples of promoters that can be used, but the invention is not limited thereto. Other promoters are known to those skilled in the art and are intended to be encompassed within the scope of the invention. The Description of all the above is incorporated herein by reference. Other useful promoters include any promoter that can be derived from a gene whose expression is aternally associated with the embryo and / or the endosperm or with early pollen formation. Preferred promoters can be used in conjunction with naturally occurring flanking transcribed or coding sequences of seed-specific genes or any other transcribed or coding sequence that is critical for seed formation and function. It may also be convenient to include some intron sequences in the promoter constructs, since the inclusion of intron sequences in the coding region can result in improved expression and specific character. Thus, it may be advantageous to link the DNA sequences to be expressed to a promoter sequence containing the first intron and exon sequences of a polypeptide that is unique to the cells / tissues of a plant and is critical to the formation and / or function of the seed. In addition, regions of a promoter can be linked to regions of a different promoter to obtain the desired activity of the promoter that results in a chimeric promoter. Synthetic promoters that regulate the expression of genes for seed development can also be used. The promoter used in the method of the invention can be an inducible promoter. An inducible promoter is a promoter that is capable of directly or indirectly activating the transcription of a DNA sequence in response to an inducer. In the absence of an inducer »the DNA sequence will not be transcribed. Typically, the protein factor that specifically binds to an inducible promoter to activate transcription is present in an inactive form which is then directly or indirectly converted to the active form by the inducer. The inducer can be a chemical agent such as a protein »metabolite (sugar» alcohol »etc.)» a growth regulator »herbicide» or a phenolic compound or a physiological stress factor imposed directly by heat »salts» toxic elements »etc . »Or indirectly through the action of a pathogen or disease agent such as a virus. An inducer can be exposed to a plant cell containing an inducible promoter by externally applying the inducer to the cell »such as by spraying» watering »heating» or similar methods. Examples of inducible promoters include the inducible 70 KD heat shock promoter of D. elanogaster (Freeling »M.» Bennet »DC» Maize DNA 1 »Ann. Rev. of Genetics 19? 297-323)» and the alcohol dehydrogenase promoter »Which is induced by ethanol (Nagao» RT and others »Miflin» BJ »Ed. Oxford Surveys of Plant Molecular and Cell Biology» Vol. 3 »page 384-438» Oxford University Press »Oxford 1986)» or the promoter Lex A that is triggered with chemical treatment and is available through Ligand Pharmaceuticals. The inducible promoter may be in an induced state during seed formation or at least for a period corresponding to the transcription of the DNA sequence of the recombinant DNA molecules. In a preferred embodiment, the promoter is one that is specific for maternal tissue such as a "tegument-specific promoter" which is expressed only in maternal tissue such as the subunit a of soybean G-congl icinin <; a'-ß-C6) »which is highly expressed in the early development of the seed in the endosperm and the embryo» as described in J.

Biol. Chem. 261: 228 (1986) »incorporated herein by reference. Another example of an inducible promoter is the chemically-inducible gene promoter sequence isolated from a 27 kd subunit of the maize gene for glutathione-S-transferase (GST II). Two of the inducers for this promoter are N »N» -diallyl-2 »Z-dichloroacetamide (name camun? Dichloramide) or = 2-clear-4- (trifluoromethyl) -5-t-thiazolcarbaxylate benzyl (common name 'flurazole ). In addition »several other potential inducers can be used with this promoter» as described in the published PCT application No. PCT / GB90 / 00110 by ICI. Another example of inducible promoter is the light-inducible chlorophyll a / b (CAB) binding protein promoter also described in PCT application published No. PCT / GB90 / 00110 by ICI. Inducible promoters have also been described in published application No. EP89 / 10388T.7 by Ciba-Geigy. In this application »several inducible promoters are identified» including the genes for PR protein »especially the genes for tobacco PR protein» such as PR-la »PR-lb» PR-lc »PR-1» PR-A »PR- S »gene for cucumber chitinase» and genes for beta-1 »3-glucanase acid and basic tobacco. There are many potential inducers for these promoters »as described in the application No. EP89 / 103888.7. The promoter can be a constitutive promoter. A constitutive promoter is a promoter that functions in all »many» or several cell types »and includes cells / tissues critical for pollen formation and / or function. An example of said constitutive promoter is 35S or preferably HP 101 of the CaMV »which have been isolated from Brassica napus. Recombinant DNA molecules containing any of the DNA sequences and promoters described herein may additionally contain marker genes for selection that encode a genetic product for selection that confers »on a plant cell» resistance to a chemical agent or factor of physiological stress "or that confers a distinguishing phenotypic characteristic to the cells" so that the plant cells transformed with the recombinant DNA molecule "can be easily selected using a selective agent. Said marker gene for selection is neomycin phosphotransferase (NPT ID) which confers resistance to kanamycin and the antibiotic G-418. Transformadae cells can be selected with this marker gene for selection by testing for the "in vitro" presence of kanamycin phosphorylation using techniques described in the literature "or by testing for the presence of the mRNA encoding the gene for NPT II by Northern blot analysis on RNA from the tissue of the transformed plant.The transformed plant cells thus selected can be induced to differentiate into plant structures Finally, it should be understood that a marker gene for selection may also be native to a plant.A recombinant DNA molecule containing any of the DNA sequences and promoters described herein may be integrated into the genome of the plant. androestéril plant or second plant »introducing first u a recombinant DNA molecule in a plant cell by any of several known methods. Preferably, "recombinant DNA molecules * are inserted into a suitable vector" and the vector used to introduce the recombinant DNA molecule into a plant cell. The use of cauliflower mosaic virus (CaMV) (Howell »SH and others» 1980 »Science 208? 265) and geminivirus (Goodman» RM »1981» J. Gen Virol. 54? 9) has been suggested as vectors »But by far the greatest success reported has been with Aqrobacterium sp. (Horsch »R.B. and others» 1985 »Science 227? 1229-1231). Methods for the use of Aqrobacte ium based on transformation systems for many different species have now been described. Generally »strains of bacteria are used they host the modified versions of the naturally occurring Ti plasmid "so that the DNA is transferred to the host plant without the subsequent formation of tumors. These methods involve the insertion "within the ts of the Ti plasmid" of the DNA to be inserted into the genome of the plant linked to a marker gene for selection to facilitate the selection of the transformed cells. Bacteria and plant tissues are cultured together to allow the transfer of introduced DNA into plant cells, and then the transformed plants are regenerated on selective media. Any number of different organs and tissues can function as targets for Aqrobacterium-mediated transformation »as specifically described for members of the Brassicaceae <; brasi caceas). These include thin layers of cells (Charest »PJ and others» 1988 »Theor, APPI, Genet, 75: 438-444)» hypocotyls (DeBlock, M. and others »1989» Plant Phvsiol .91? 694-701) »disks foliar (Feldman »KA and Marks» MD »1986» Plant Sci. 47? 63-69) »stems (Fry J. and others» 1987 »Plant Cell Rep. 6? 321-325>» cotyledons (Moloney MM and others »1989» Plant Cell Repts 8: 238-242) and embroids (Neuhaus »G. and others» 1987 »Theor, APPI, Genet 75: 30-36), however it is understood that it may be convenient in some crops selecting a different tissue or transformation method It may be useful to generate several individual transformed plants with some recombinant construction to recover plants free of some position ct.It may also be preferable to select plants that contain more than one copy of the introduced recombinant DNA molecule »So that high levels of expression of the recombinant molecule are obtained. all that have been used to introduce recombinant molecules into plant cells involve mechanical means »such as direct DNA uptake» liposomes »electroporation (Guerche» P. and others »1987» Plant Science 52: 111-116) and icroinjection (Neuhaus » G. and others »1987» Theo. APPI. Genet 75: 30-36). Considerable attention has also been given to the possibility of using microprojects and a gun or other devices to force small metal particles coated with DNA into cells (Klein »T.M. and others» 1987 »Nature 327: 70-73). It is contemplated in some of the embodiments of the method of the invention that a plant cell be transformed with a recombinant DNA molecule containing at least two DNA sequences or that it be transformed with more than one recombinant DNA molecule. The DNA sequences or the recombinant DNA molecules in said modalities »may be physically bound while being in the same vector» or they may be physically separated in different vectors. A cell can be transformed simultaneously with more than one vector "as long as each vector has a marker gene for single selection. Alternatively, "a cell can be transformed with more than one vector" by sequentially allowing an intermediate regeneration step after the transformation with the first vector. In addition »it may be possible to perform a sexual crossing between individual plants or plant lines containing different DNA sequences or recombinant DNA molecules? preferably "the DNA sequences or recombinant molecules are linked or located on the same chromosome" by which plants containing DNA sequences or recombinant DNA molecules can be selected from the progeny of the crossing. The expression of recombinant DNA molecules containing the DNA sequences and promoters described herein can be monitored in transformed plant cells using Northern blot techniques and / or Southern blot techniques known to those skilled in the art. As indicated above, it may be convenient to produce plant lines that are ho-zygotic for a particular gene. In some species, this is best achieved easily through the use of anther culture or the cultivation of isolated microspores. This is especially true for the cultivation of oilseeds Brassica napus (Keller and Armstrong »Z. flanzenzucht 80: 100-108» 1978). By using these techniques it is possible to produce a haploid line carrying the inserted gene and then duplicate the chromosome number spontaneously or by the use of colchicine. This results in a plant that is homozygous for the inserted gene which can easily be tested if the inserted gene carries a marker gene for proper selection to detect plants bearing said gene. Alternately »plants can be selfed» resulting in the production of a seed mixture consisting » in the simplest case »of three types» ho ocigótico (25%) »heterozygous» (50%) and null (25%) for the inserted gene. Although it is relatively easy to achieve null plants from those containing the gene, it is possible in practice to select homozygous heterozygous plants by Southern blat analysis, in which case special attention is n to loading exactly equivalent amounts of DNA from the population. mixed "and selecting the heterozygotes by means of the intensity of the signal from a specific probe for the inserted gene. It is advisable to verify the results of the Southern blat analysis by "allowing each independent transformant to self-fertilize" since additional evidence of homacigocity can be obtained by the simple fact that if the plant was homozygous for the inserted gene all the subsequent seed plants Authenticated seed will contain the gene "whereas if the plant was heterozygous for that gene" the generation obtained from the self-fertilized seed will contain null plants. Therefore »using simple self-fertilization» it is possible to easily select lines of homozygous plants that can also be confirmed by Southern blot analysis. Several different ways of producing the hormone molecule specifically synchronized with the development of pollen or with fertilization can be envisaged. In all procedures »at least one step in the production of the hormone molecule has occurred specifically within a tissue involved in pollination and fertilization at a time shortly before it to induce the plant to continue with the formation of the fruit. Any number of genes can be used to carry out the method and methods of the invention "provided that the simultaneous production of two or more enzymatic or synthetic activities specifically in the plant leads to the enhancement of the synchronized gibberellin prior to fertilization and the development of the seed. This implies that any intermediate can be used in gibberellin biosynthesis "as well as enzymes that catalyze these reactions or even receptors activated by the presence of gibberellin. In still mode "the gene may be an antisense oligonucleotide" which will interfere with »and prevent the transcription of» an enzyme that degrades or inhibits gibberellin. As can be seen, various combinations of structural genes and promoters specific to the development of pollen are contemplated within the scope of the invention. A highly suitable seedless system "is one in which fully fertile Fl seed is developed" and which can be developed in plants that produce only seedless fruits. This system is economically favorable "because for each cross-pollination" a large number of seedless fruits is obtained: the number of seeds Fl of a cross X corresponds to the number of fruits produced in a plant Fl. Also incorporated in this scheme are the advantages of developing a hybrid crop »including the most valuable combination of hybrid characteristics and vigor. This is achieved in the same manner as described above, except that the gene for hormone is expressed from an inducible or repressible promoter. This will allow the generation of progenitor lines that will carry "but will not express" the gene. The hybrid Fl between the two will thus also have the gene "and after the application of an agent that will induce the expression" will be tenocular pair. With a repressible promoter, the parents will express the gene, but this will be repressed in the female progenitor used for the production of the seed. The use of specific promoters of the temperature »or suppressible promoters» is also useful to generate F1 hybrids. The essence of the parthenocarpic phenotype is the expression of gibberellin near or at the time of pollination »which should induce the signal of that the production of the seed has been initiated. The formation of the seed and the early development of it is responsible for the formation of the fruit and its normal maturation in pepper and other fruits and vegetables. Another approach is to modify the receptor for GA to increase sensitivity to endogenous GA levels. The product of the SPY1 gene is involved in signal transduction for GA »that is» a receptor for GA. A combination of a GA-modified receptor and fruit-specific promoters can be used to modify the fruit phenotype analogous to the use of genes that result in higher levels of bioactive GA.

Claims (26)

N0V5PP PE THE INVENCI N CLAIMS

1. - An expression construct for the production of tranegenic parthenocarpic plants »characterized in that it comprises: a recombinant gene that encodes the expression of a phytohormone» precursor of the same »or enzyme that intervenes in the biosynthesis thereof» where said hormone promotes the formation and / or the development of the fruit »and a specific promoter of the pollen linked operably to this gene.

2. The construction of expression according to claim 1 »characterized in that said phytohormone is gibberellin.

3. The expression construction according to claim 2 »characterized in that said phytohormone is gibberellin oxidase.

4. The genetic construction according to claim 1 »characterized in that said gene is a gene for ear and anther 1.

5. The genetic construct according to claim 1» characterized in that said gene is a gene for ear and anthers 2.

The expression construction according to claim 1, characterized in that said plant is a pepper plant.

7. The expression construct according to claim 1 »characterized in that said promoter is an inducible promoter.

8. A nucleic acid vector "characterized in that it comprises the expression construct according to claim 1.

9. The vector according to claim 8» characterized in that said vector is a cloning vector.

10.- The vector in accordance with the claim 8 »characterized in that said vector is an expression vector.

11. The vector according to claim 8 »characterized in that it also comprises a marker gene for the selection of transformed cells.

12. The vector according to the claim 11 »characterized in that said marker gene is selected from the group consisting of a gene for ampicillin resistance» a gene for tetracycline resistance and a gene for hygromycin resistance.

13.- The vector in accordance with the claim 8 »characterized in that it further comprises a polyadenylation signal.

14. A pracaritic or eukaryotic host cell, characterized in that it is transformed with the nucleic acid vector according to claim 7.

15.- A transgenic plant »characterized in that it comprises a plant cell or an ancestor of said plant cell» which have been transformed with the vector according to claim 8.

16.- A parthenocarpic fruit produced by a plant that contains a DNA sequence that codes for a recombinant gene that codes for a phytohormone that initiates the formation or development of the fruit »a precursor of said hormone» to enzymes involved in the synthesis of said hormone »characterized in that said gene is operably linked to a specific pollen promoter» so that the expression occurs before pollination.

17. A method for producing parthenocarpic fruits, characterized in that it comprises: transforming an angiosperm plant cell with a DNA sequence that codes for a phytohormone that intervenes in the beginning of fruit formation "to a precursor of said hormone or an enzyme which intervenes in the synthesis of said hormone operably linked to a specific pollen promoter; and generating a plant from said transformed cell; where said plant is parthenocarpic and the formation of the fruit begins without pollination.

18. The method according to claim 17 »characterized in that said plant is selected from the group consisting of a melon plant» a pepper plant and a tomato plant.

19. The method according to claim 17 »characterized in that said DNA sequence codes for gibberellin oxidase.

20. The method according to claim 19 »characterized in that said promoter is selected from the group consisting of: a constitutive promoter »an inducible promoter and a maternal tissue promoter.

21. A method for producing a hybrid parthenocarpic plant "characterized in that said method comprises: pollinating a pragenitara plant that has been transformed" or whose ancestor has been transformed with a DNA sequence that codes for a genetic product for phytohormone operably linked to a inducible promoter? with a second pragenitara plant that has been transformed »or whose ancestor has been transformed with a similar gene» said sequence of DNA operably linked to an inducible promoter »so that after the application of the inducer» the plant will be parthenocarpic.

22.- A method to produce parthenocarpic fruits and vegetables »characterized because it comprises? transform a plant cell or an ancestor of it with a DNA sequence that will enhance the expression of gibberellin »said sequence being regulated by a specific pollen promoter? and generating a parthenocarpic plant from said cell.

23. The method according to claim 22 »characterized in that said DNA sequence is a structural gene.

24. - The method according to claim 23 »characterized in that said gene is a gibberellin receptor.

25. The method of compliance with claim 23 »characterized in that said gene codes on the expression of gibberellin oxidase. 26.- A method to produce tenacarpal fruits »characterized because it comprises? transforming an angiosperm plant cell with a DNA sequence that codes for a phytohormone involved in the start of fruit formation, or a precursor of said hormone or an enzyme that intervenes in the synthesis of said hormone operably linked to a specific promoter of the pollen wherein said hormone is au ina »and generate a plant from said transformed cell; where said plant is parthenocardic and the formation of the fruit begins without pollination. ftSMIN PE INVENTION The invention describes a transgenic method to produce parthenocarpic fruits or fruits with a reduced number of seeds »implies the temporary expression of a phytohormone» precursor or a gene »so that the activity of gibberellin or other similar hormonal activity is enhanced at the beginning of the fruit formation activity; the gene is operably linked to a regulatory promoter "so that expression is synchronized before pollen development or fertilization; the expression of the hormone causes fruit development in the absence of fertilization; the method also results in a fruit that has small seeds or very few seeds; The invention also includes constructs, vectors and transgenic methods for the production of parthenocarpic plants. MG / amm * ehp * mmr. P98-1210F.