MXPA98008802A - Genetic constructions and methods to produce fruits with very little seed or with reduced size seeds - Google Patents

Abstract

The invention describes a transgenic method to produce fruits with seeds of diminished size or very few seeds, or fruits with a reduced number of seeds, implies the temporary expression of a cytotoxic gene or combination of genes destined to stop the development of the seed to a time sufficiently after the pollination, so that the development and maturation of the fruit are normal, but quite early in the development of the seed so that the maturation of the same is reduced to the minimum per se. The invention also includes transgenic constructions, vectors and methods for producing fruits with seeds of diminished size, or very few seeds in plants that produce the same seeds.

Description

GENETIC CONSTRUCTIONS AND METHODS TO PRODUCE FRUITS WITH VERY LITTLE SEED OR REDUCED SIZE SEEDS FIELD OF THE INVENTION This invention relates generally to the field of plant molecular biology and in particular to plants, seeds and transgenic tissues that have been genetically modified to create plants that when pollinated, produce fruits and vegetables without seed.

BACKGROUND OF THE INVENTION 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, since the seed cavity is absent or greatly reduced. Several advances have been made in this field, usually with the topical application of hormones or with highly complex reproductive procedures that result in a triple genotype. For example, the topical application of gibberellins has been used for a long time for the production of seedless es. This method usually requires a sprinkling procedure that is problematic, depends on the climate and must be regulated in a timely manner so that it occurs immediately after pollination. Another method requires a complex breeding procedure for the production of seedless watermelons. The seedless condition of the watermelon is almost always the result of the presence of three homologous supplements per cell instead of the normal two, known as triploid. The development of the seed is stopped due to the unbalanced constitution of the genome to the embryo and the endosper, as well as the meiosis and irregular gamete formation. The abnormal formation of the embryo causes the cessation of the normal ovular development of a seed in an initial stage. Seedless watermelons typically contain small, edible white eggs similar to those of unripe cucumbers. These triploid seeds are produced by crossing male diploid (2X) lines containing 22 chromosomes per cell with female tetraploid (4X) lines containing 44 chromosomes per cell. The triploid genotype is produced by a 4n-2n cross, the 3n seed is "normal" and is planted by growth. Triploid plants are true Fl hybrids, so their production depends on the development of diploid and tetraploid progenitor lines. For the large-scale commercial production of triploid seeds, tetraploid and diploid progenitor lines are planted in mixed plots and allowed to cross-pollinate. The triploid seed is produced only in melons on tetraploid plants that are fertilized with diploid pollen. The 3n watermelon production fields have diploid pollinators. Some 3n plants produce very little pollen, and diploid pollen achieves inefficient pollination. The development of the seed is stopped after fertilization due to the general imbalance. In this way, an adequate supply of diploid and tetraploid seeds should be available to produce large mixed crops. The main limitation to producing this type of seedless watermelon has been the difficulty associated with the production of sufficient seeds for the 4X tetraploid progenitor lines. Traditionally this is achieved by the application of colchicine. Colchicine inhibits the formation of the itotic beam, which leads to cells with several numbers of chromosomes. The tetraploids are produced by applying one drop per day of 0.2 to 0.4% aqueous colchicine at the apices of the diploid seedling. Commonly this treatment with colchicine results in the death of the seedling or seed, or produces aneuploids (cells with extra or missing chromosomes or polyploids). Once a desirable cultivar is identified and the number of chromosomes is doubled, it is self-pollinated to form a suitable seed. However, tetraploid seed has been very difficult to produce in commercially useful large quantities, due in large part to the fact that tetraploids exhibit a high degree of self-sterility. In this way, very few melons develop in a field of tetraploid plants. Seed production is more difficult in tetraploid progors produced in each self-pollinated melon. Typically ten or more years are required to increase the seed of a new tetraploid line to commercially acceptable numbers. The patent of E.U.A. No. 5,007,198 to Dennis Cray et al., Describes a method for increasing the production of the tetraploid progor line for watermelon by overcoming the sterility of the tetraploid cell and allowing cloning of the cultivar using unique tissue culture techniques. Generally, the method uses a tissue culture propagation method to "clone" the 4n parents by using the seedling meristem as the starting tissue for the propagation of an apex of watermelon in a petri dish filled with nutrients and regulators of growth, resulting in the outbreak of up to 15 single-apex shoots. The rods are used in turn to sprout even more crops resulting in a geometric increase. A transg method to create seedless fruits is described in the world patent W091 / 09957. This includes a highly complex recombination excision system called "CRE-LOX". Generally, the CRE gene product produces a porotein recombinase 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 Bacillus amyloiquefaciens. Generally, the female seed has a specific tegument (SC) promoter: CRE: terminator and the male seed has a SC promoter: barnase gene :: lox:: barnase gene: erminator. There are no changes of phenotype in the two parents when they are propagated and in the field of seed production the parent of the female seed has a normal set of seeds because both genes are not working at the same time (the tegument is only maternal tissue) . However, in generation Fl the maternal tissue will contain both genes. In this configuration, the constitution of the integument (plant Fl, seeds F2) is SC promoter: gene barnase:: -:: gene bamasa: terminator, where :: - :: represents the point where the lox gene is excised making a Functional toxin gene specific for the tegument. In theory, if the tegument is decomposed the development of the seed will be stopped due to the lack of nutrient flow towards the embryo. Experimental data are not described and the success of the proposed protocol in the development of real seedless watermelons is not shown anywhere.

In this way, it can be observed from the above that the technique requires production protocols for fruits and vegetables without seeds, which 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 production of seedless fruits and vegetables. It is also another object of the invention to provide inbred progenitor lines that can be reproduced cross-wise resulting in a plant Fl that will not produce seeds or 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 in 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 maintenance replication and transformation of the plant. Other objects of the invention will become apparent from the following description of the invention.

BRIEF DESCRIPTION OF THE INVENTION The invention comprises the temporal expression of a cytotoxic gene or combination of genes focused on stopping the development of the seed at a time sufficiently after pollination for the development and maturation of the fruit to be normal, but early enough in the development of the seed so that the maturation of the seed is reduced to the minimum per se. In one embodiment, the invention comprises a first DNA sequence that encodes a first gene product that is capable of making a nontoxic, cytotoxic substance for the cell of a plant that is critical to the formation and / or function of the seed, and a second DNA sequence encodes a second gene product that is a non-toxic substance, or encodes a second gene product that is capable of converting an endogenous substance for a plant cell into a non-toxic substance, which can be converted by said first product of gene in a cytotoxic substance. The combination of two genes makes possible the production of hybrid plants (Fl) that will produce fruits or vegetables (F2) without seed. Transgenic homozygous progenitor lines are maintained with each parent containing either the first or second recombinant DNA sequence operably linked to a seed-specific promoter. The two sequences are combined only when the parent lines are crossed and result in a fully functional cytotoxic gene, whose expression is regulated with the development of the seed. Preferably, the first DNA sequence encodes indolacetamide hydrolase (Ia H), which converts indolacetamide into the native plant auxin indoleacetic acid (otherwise known as auxin gene 2) the second DNA sequence encodes indolacetamide synthetase (lamS) which converts tryptophan to indolacetamide (otherwise known as auxin gene 1) and the first and second promoters are seed-specific promoters. The overproduction of auxin in a tissue-specific manner can result in localized toxicity to that tissue. The overproduction of auxin bound to a specific pollen promoter has previously been used to produce sterile male plants, see U.S. Pat. 5,426,041 to Fabijans and incorporated herein by reference. In another embodiment of the invention, the first and second cytotoxic recombinant DNA sequences can be transformed into the same plant which can then be reproduced or vegetatively grown to produce other genetically identical plants.

DETAILED DESCRIPTION OF THE INVENTION In the following description, a number of terms are used extensively. The following definitions are provided to remove ambiguities in the intention or scope of their use in the description and claims, as well as to facilitate understanding of the invention. As used herein, the term "fruit" shall include any angiosperm plant that has its pollen and egg-producing organs in flowers; the ovules are enclosed in an ovary, and after fertilization, each ovule develops to form a seed while the ovary expands to form a fruit. Any fruit of this type that one wishes to produce without seed is covered by this definition. Also included are additional food sources traditionally considered vegetables but produced in this manner, such as tomatoes, peppers and the like. A cytotoxic protein is one that disrupts the normal functions of the cell. 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 specific seed promoter is any promoter capable of regulating the temporal expression at a time sufficiently after pollination so that the development and maturation of the fruit are normal, but that it is early enough in the development of the seed for the maturation of the seed. it is reduced to the minimum. Such promoters such as those mentioned herein include, but are not limited to, inducible promoters, seed-specific promoters of maternal, paternal or hybrid origin, or any other promoter associated with a gene involved in the production or development of the seed. The term "expression" refers to the biosynthesis of a gene product. Expression of the structural gene includes transcription of the structural gene into mRNA and then translation of the mRNA into one or more polypeptides. A cloning vector is a DNA molecule such as a plasmid, cosmid 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 the introduced DNA sequences can be inserted in a determinable manner without loss of the essential biological function of the vector, as well as a marker gene. which is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide resistance to tetracycline or resistance to ampicillin. An expression vector is a DNA molecule that comprises a gene that is expressed in a host cell.

Typically, the expression of the gene is brought under the control of certain regulatory elements including 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 prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes prokaryotic or eukaryotic cells that have been genetically engineered to contain the clone 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. The plant tissue includes differentiated and undifferentiated tissues or plants, including but not limited to: roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various cell forms and cultures such as individual cells, protoplasts, embryos and callosum tissue . The plant tissue can be in a plant or in an organ, tissue or cell culture.

General Recombinant 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 alternative files for each stage of the global procedures. 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, promoter elements and elements towards the xtreme 5 'used. Those skilled in the art are capable of selecting and using suitable alternatives to achieve functionality. Culture 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 containing and expressing desired genes can be obtained 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. The selection of the truncated promoter and the selection of the structural cytotoxic gene 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 of 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 an antibiotic resistance marker to provide growth and selection of the expression vector in a bacterial host; (2) DNA elements that control the initiation of transcription such as a promoter; (3) DNA elements that control the processing of transcripts such as transcription termination / polyadenylation sequence; and (4) a reporter gene that is operably linked to the DNA elements to control the initiation of transcription. Useful reporter genes include β-glucuronidase, β-galactosidase, chloraf-en-acetyl transferase, luciferase and the like. Preferably, the reporter gene is either β-glucuronidase (GUS) or luciferase. General descriptions of plant expression vectors and reporter genes can be found in Gruber, et al., "Vertors for Plant Transformation, in Methods in Plant Molecular Biology &Biotechnology" in Glich et al., (Eds. Pp. 89-119, CRC Press, 1993). Furthermore, expression vectors for GUS and gene cassettes for GUS are available from Clone Tech Laboratories, Inc., Palo Alto, California, while luciferase expression vectors and gene cassettes for luciferase 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 growing plant tissues are provided for example by Maki et al., "Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology & Biotechnology, Glich et al. (Eds. Pp. 67-88 CRS Press, 1993); and by Phillips et al., "Cell-Tissue Culture and In-Vitro Manipulation" in Corn & Corn Improvement, 3a. Edition, Sprague et al. (Eds. Pp. 345, 387) American Society of Agrono and Inc. and others 1988. Methods for introducing expression vectors into plant tissue include direct infection or co-cultivation of the plant cell with Agrobacterium tumefaciens , Horsch et al., Science, 227: 1229 (1985). The vector system descriptions of a robacterium and methods for agrobacterium-mediated gene transfer are provided by Gruber et al., Supra. Useful methods include, but are not limited to, expression vectors that are introduced into plant tissues using a direct gene transfer method such as microprojectile-mediated release, DNA injection, electroporation and the like.

Most preferably, expression vectors are introduced into tissue advantages using the release of microprojectile medium with biolistic device. The invention comprises the use of these types of transformation methods to generate transgenic plants bearing a seedless fruit. A construct comprising a cytotoxic gene operably linked to a specific seed promoter is introduced into said plant. The cytotoxic gene can be a single gene product or a combination of genes. In a preferred embodiment, the invention comprises the use of a pair of genes which, when expressed together, create a toxic gene product. This can allow the production of seeds or hybrid plants, once the transgenic inbred progenitor lines have been established. For this embodiment, the invention comprises a first DNA sequence encoding a first gene product, which is capable of making a nontoxic, cytotoxic substance for a plant cell that is critical to the formation and / or function of the seed , and a second DNA sequence encodes a second gene product that is a non-toxic substance, or encodes a second gene product that is capable of converting an endogenous substance for a plant cell into a non-toxic substance. It is desirable to have the first DNA sequence and the second DNA sequence in the ho ocigotic state, which may require more than one transformation event to create each parent line, requiring transformation with a first and second recombinant DNA molecule both of which which encode the same gene product. Preferably, the first recombinant DNA molecule and the second recombinant DNA molecule are located on opposite chromatids of the same pair of chromosomes and most preferably on opposite chromatids of the same pair of chromosomes in the same genetic locus, whereby the segregation of the first and second recombinant DNA molecules occur during meiosis, and the opportunity for recombination of the first and second recombinant DNA molecules for the same chromatid during eicotic crosslinking is substantially reduced. The methods of the invention described herein may be applicable to any species of seed-bearing plant, whose fruit or vegetable is edible or whose interior it is desirable to make without seed for reasons of discomfort or other reasons. Fruits and plant plants that can be made seedless according to the methods of the invention include, but are not limited to, melons such as common melon, melon drop of honey and watermelon, Chinese melon, berries such as strawberries and blueberries, peppers such as green peppers, red peppers, yellow peppers, tomatoes, oranges, plums, alfalfa, squash, eggplant, sweet corn, peas, cotton, avocados, mangoes, papaya, nectarines , apples, grapefruit, lemons, limes, tangerines, pears and peaches. The methods of the invention will be illustrated below with reference to particular embodiments. As mentioned hereinabove, the first DNA sequence can encode a first gene product that is capable of making a nontoxic, cytotoxic substance for a plant cell that is critical for the formation and / or function of the seeds , and the second DNA sequence can encode a second gene product that is the non-toxic substance or encode a second gene product that is capable of converting an endogenous substance for a plant cell into said non-toxic substance. A cell and / or tissue of a plant that is critical to the formation and / or function of the seed includes cells and / or tissues that are instrumental in development, or seeds - including cells and / or tissues from which ee develop seeds, cells and / or tissues that are part of the tegument, endospore or embryo or other female structure in which the seed is developed (eg, ovules), but without interrupting the maturation of the ovary to form a fruit or vegetable . The first DNA sequence can be any identifiable DNA sequence that encodes gene products that are capable of making a nontoxic, cytotoxic substance to the cell of a plant that is critical to the formation and / or function of the seed. Examples of said DNA sequence include a DNA sequence encoding indolacetamide hydrolase (Ia H) (auxin gene 2) that converts naphthalene acetamide into the plant growth regulator alpha naphthalene acetic acid (NAA) (auxin gene 2) which It converts indole acetamide into indole acetic acid (IAA) which is a regulator of plant growth. A source of enzyme Ia H is the bacterium Agrobacterium tumefaciens (Inze, D. et al., 1984, Mol. Gen. Genet. 194: 265-74 and Koncz, C. and Schell, J., 1986, 204: 383-396 derived from plasmid pPCV 311). Another source of an enzyme that is genetically equivalent to IamH is the gene coding for indole acetamide hydrolase from Pseudomonas suvastanoi (Follín et al. (1985) Mol.Gen.Genet., 201: 178-185). Another source is the patent of E.U.A. No. 5,426,041. Other cytotoxic genes include DAM methylase, diphtheria toxin or any other protein that disrupts the normal function of the cell. The first DNA sequence can also encode a gene product that is capable of making a non-toxic substance that is a protoxin, cytotoxic to the cell of a plant that is critical to the formation and / or function of the seed. A protoxin has been identified that is inactive against plants, but that after enzymatic conversion becomes cytotoxic. (Dotson, S.B. and G.M. Kishore, Isolation of a Dominant Lethal Gene with Potential Uses in Plants in The Genetic Dissection of Plant Cell Processes 1991).

The second DNA sequence can encode a second gene product that is the non-toxic substance, or encode a second gene product that converts a substance that is endogenous to a plant cell in the non-toxic substance. For example, a cell can contain a DNA sequence encoding IamH (auxin gene 2) (which converts indolacetamide to cytotoxic levels of indoleacetic acid), and a DNA sequence encoding S (auxin gene 1). IamS converts tryptophan which is generally endogenous to plant cells, in indolacetamide, which in turn is converted by Ia Ia into cytotoxic levels of indoleacetic acid. A source of the IamS enzyme is gene 1 of T-DNA of the bacterium Agrobacterium tumefaciens (Inze, D. et al., 1984, Mol.Gen.Genet, 194: 265-74). Another source of an enzyme that is functionally equivalent to lamS is the gene encoding tryptophan 2-mono-oxygenase from Pseudomonas savastanoi (Follín et al., (1985) Mol. Gen. Genet, 201: 178-185). Another source is the patent of E.U.A. No. 5,426,041. The DNA sequence can also encode non-toxic substances such as the aforementioned protoxin. In a single gene modality, the cytotoxic gene used is selected from a group of genes encoding products that disrupt the normal functioning of the cells. There are many proteins that are toxic to cells when they are expressed in an unnatural situation. Examples include the genes for the restriction enzyme EcoRI [Barnes and Riñe, Proc. Natl. Acad. Sci. USA 82: 1354-1358 (1985)], diphtheria toxin A [Yamaizumi et al., Cell 15: 245-250 (1987)], streptavidin [Sano and Cantor, Proc. Natl. Acad. Sci. USA 87: 142-146 (1990)] and barnase [Paddon and Hartley, Gene 53: 11-19 (1987)]. The promoters used in the methods of the invention may be a seed specific promoter, an inducible promoter or a constitutive promoter. The seed specific promoter used is selected from the group of known promoters because they direct expression in the embryo and / or the endosperm of the developing seed, most desirably in the endosperm. A set of examples of seed-specific promoters includes, but is not limited to, promoters of seed storage proteins. Seed storage proteins are strictly regulated, being expressed almost exclusively in seeds in a highly tissue-specific and stage-specific form [Higgins et al., Ann. Rev. Plant Physiol., 35: 191-221 (1984); Goldberg et al., Cell 56: 149-160 (1989)]. Similarly, different seed storage proteins can be expressed at different stages of seed development and in different parts of the seed. Other examples include maternal tissue promoters such as tegument, pericarp and ovule. There are numerous examples of seed-specific expression of seed storage protein genes in transgenic dicotyledonous plants. These include genes of dicotyledonous plants for bean β-phaseolin [Sengupta-Goplalan and others, Proc. Natl. Acad. Sci. USA 82: 3320-3324 (1985) and Hoffman et al., Plant Mol. Biol. 11: 717-729 (1988)], bean lectin CVoelker et al., EMBO J 6: 3571-3577 (1987)], soybean lectin [Ocamuro et al., Proc. Natl. Acad. Sci. USA 83"8240-8344 (1986)], inhibitor of kunitz trypsin from soybean [Perez-Grau and Goldberg Plant Cell 1: 1095-1109 (1989)], soybean-conglycinin [Beachy et al., EMBO J 4 : 3047-3053 (1985), Barker et al., Proc. Nati Acad. Sci. 85458-462 (1988), Chen et al. 'EMBO J 7: 297-302 (1988), Chen and others Dev. Genet. 11: 112-122 (1989), Naito et al., Plant Mol. Biol. 11: 683-695 (1988)], vi pea pea [Higgins et al., Plant Mol. Biol. 11: 109-123 (1988)], chickpea convicilina (Newbigin et al., Planta 180: 461 (1990)], peas' legumin [Shirsat et al., MOI.

Gene ti cs 215: 326 (1989)], rape napkin [Radke et al., The ror. Appl. Genet 75: 685-694 (1988)], as well as moneto ti plant genes such as for corn 15 kd zein [Hoffman et al., EMBO J 6: 3213-3221 (1987)], barley ß-hordein [Marris and others, Plant Mol. Biol. 10: 3259-366 (1988), and wheat glutenin CColot et al., EMBO J 6: 3559-3564 (1987)]. Moreover, promoters of seed-specific genes operably linked to heterologous coding regions in chimeric gene constructs also maintain their pattern of temporal and spatial expression in transgenic plants. Such examples include the gene promoter from the 2S seed storage protein of Arabidopsis thaliana to express enkephalin peptides in seeds of rabidopsis and Brassica napus [Vandekerckhove et al., Bio / Technology 7: 929-932 (1989)], promoters of bean lectin and bean β-phaseolin to express luciferase CRiggs et al., Plant Sci. 63: 47-57 (19889)], and wheat glutenin promoters to express chlorafmenicol acetyltransferase [Colot et al., EMBO J 6: 3559-3564 (1987)]. The promoters highly expressed in the development of the endosperm are very effective in this application. Of particular interest is the promoter of the α subunit of the soy β-conglycinin gene [Walling et al., Proc. Natl. Acad. Sci.

USA 83: 2123-2127 (1986)] which is expressed at the beginning of the development of the seed in the endosperm and the embryo. A seed-specific promoter is the DNA sequence that regulates the expression of a DNA sequence selectively in the cells / tissues of a plant critical for the formation and / or function of the seed, and / or which limits the expression of said sequence of DNA to the period of seed formation in the plant. Any specific identifiable seed or embryo promoter can be used in the methods of the present invention. Other promoters that are seed or embryo specific and that are useful in the method of the invention include the phaseolin promoter described in the US patent. No. 5,504,200 to Mycogen, which is incorporated herein by reference. This patent describes the gene sequence and regulatory regions for phaseolin, a protein isolated from P. vulgaris. The phaseolin protein is highly regulated and the gene encoding the protein is expressed only while the seed is in development within the pod, and only in tissues involved in the generation of the seed. Another promoter is the napin promoter described in the U.S. patent. No. 5,110,728 to Calgene, which is also incorporated herein by reference. The patent describes the use of the napin promoter to direct the expression of an acyl carrier protein to the plant tissue to increase seed oil production. Other promoters include the carrot DC3 promoter which is specific for premature to intermediate embryo and is described in Plant Physiology, Oct. 1992 100 (2) p.576-581, "Hormonal and Environmental Regulation of the Carrot Lea-class Gene Dc 3 Another is described in Plant Mol. Biol., April 1992, 6/18) p.1049-106, "Transeriptional Regulation of a Seed Specific Carrot Gene, DC 8." Other useful promoters include any promoter that can be derived from a gene whose expression is maternally associated with the embryo and / or endosperm or late pollen formation.Preferred promoters can be used in conjunction with flanking transcriptional or coding sequences that naturally occur from seed-specific genes or with any another coding or transcribed sequence that is critical for seed formation and / or function It may also be desirable to include certain intron sequences in the promoter constructs, since the inclusion of intron sequences in the coding region can result in improved expression and specificity. In this way, it may be advantageous to bind the DNA sequences that will 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 critical plant for the formation and / or function of the seeds. In addition, the regions of a promoter can be linked to regions of a different promoter to obtain the desired promoter activity resulting in a chimeric promoter. Synthetic promoters that regulate gene expression for seed development can also be used. The first promoter or the second 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 that is then directly or indirectly converted to the active form by an 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 from D. melanogaster (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 et al., Miflin, BJ, Ed. Oxford Surveys of Plant Molecular and Cell Biology, Vol. 3, pp. 384-438, Oxford University Press, Oxford 1986), or the promoter Lex A which is unchained with chemical treatment and is available through Ligand Ph rmaceuticals. 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 to the integument, which is expressed only in maternal tissue such as the α subunit of soybean β-conglycinin (a'-β-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: 9228 (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-2,2-dichloroacetamide (common name: dichloro ida) or = 2-chloro-4- (trifluo romethyl) -5-thiazolecarboxylate 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 an inducible promoter is the light-inducible chlorophyll a / b (CAB) binding protein promoter, also described in the published PCT application No.

PCT / GB90 / 00110 by ICI. Inducible promoters have also been described in published application No. EP89 / 103888.7 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- a, PR-lb, PR-lc, PR-1, PR-A, PR -S, the gene for cucumber chitinase, and the genes for beta-l, 3-glucanase, acid and basic tobacco. There are many potential inducers for these promoters, as described in the application No. EP89 / 103888.7.

The first or second promoter can be a constitutive promoter. A constitutive promoter is a promoter that functions in all, many, or several types of cells, and includes cells / tissues critical for the formation and / or function of pollen. An example of said constitutive promoter is 35S or Super mass or HP 101 of CaMV, which has been isolated from Brassica napus. Recombinant DNA molecules that contain 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, in 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 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 II), which confers resistance to kanamycin and the antibiotic G-418. Transformed cells with this marker gene can be selected for selection by testing for the presence, in vitro, 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 analysis. blot in RNA from the tissue of the transformed plant. It can be induced that the transformed plant cells thus selected differentiate into plant structures that ultimately produce whole plants. 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, can be integrated into the genome of the male sterile plant or second plant, by first introducing a recombinant DNA molecule into a plant cell by any of several methods known. Preferably, the recombinant DNA molecules are inserted into a suitable vector, and the vector is used to introduce the recombinant DNA molecule into a plant cell. The use of cauliflower mosaic virus (CaMV) (Howell, SH et al., 1980, Science 208: 1265) and geminivirus (Goodman, RM, 1981, J. Gen Virol. 54: 9) as vectors has been suggested. , but by far the greatest success reported has been with Agrobacterium sp. (Horsch, R.B. and others, 1985, Science 227: 1229-1231). Methods for the use of Agrobacterium based on transformation systems for many different species have now been described. Generally, strains of bacteria harboring the modified versions of the naturally occurring Ti plasmid are used, so that the DNA is transferred to the host plant without the subsequent formation of tumors. These methods involve the insertion, within the limits 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 transfer of the 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 of the Agrobacterium-mediated transformation, as specifically described for members of the Brassicaceae (Brassicaceae). These include thin layers of cells (Charest, P.J. et al., 1988, Theor. Appl. Genet. 75: 438-444), hypocotyls (DeBlock, M. et al., 1989, Plant Phvsiol. 91: 694-701), forliary disks (Feld an, KA and Marks, MD, 1986, Plant Sci. 47: 63-69 ), stems (Fry J. et al., 1987, Plant Cell Repts. 6: 321-325), cotyledons (Moloney MM et al., 1989, Plant Cell Repts. 8: 238-242) and broids (Neuhaus, G. others, 1987, Theor, Appl. Genet, 75: 30-36). However, it is understood that it may be desirable in some crops to select 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 effect. It may also be preferable to select plants containing more than one copy of the introduced recombinant DNA molecule, so that high levels of expression of the recombinant molecule are obtained.

Other methods that have been used to introduce recombinant molecules into plant cells involve mechanical means, such as direct uptake of DNA, liposomes, electroporation (Guerche, P. et al., 1987, Plant Science 52: 111-116) and microinjection (Neuhaus). , G. et al., 1987, Theor, Appl. Genet, 75: 30-36). Considerable attention has also been given to the possibility of using microprojectiles and a gun or other devices to force small metal particles coated with DNA into cells (Klein, T.M. et al., 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 embodiments 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, sequentially allowing an intermediate regeneration step after 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, so plants containing DNA sequences or recombinant DNA molecules can then 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 achieved rather 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 be easily tested if the inserted gene carries a marker gene for proper selection to detect plants bearing said gene. Alternatively, the plants can be self-fertilized, resulting in the production of a seed mixture consisting of, in the simplest case, of three types, homozygous (25%), homozygous, (50%) and null (25%) for the inserted gene. Although it is relatively easy to achieve null plants from those that contain the gene, it is possible in practice to select homozygous heterozygous plants by means of Southern blot analysis, in which case special attention is given to loading exactly equivalent amounts of DNA from the population mixed, and selecting heterozygotes by means of signal intensity from a specific probe for the inserted gene. It is advisable to verify the results of the Southern blot analysis, allowing each independent transformant to self-fertilize, since additional evidence of homozygosity can be obtained by the simple fact that if the plant was homozygous for the inserted gene, all subsequent plants of the seed self-fertilized seed will contain the gene, while 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 homozygous plant lines that can also be confirmed by Southern blot analysis. Various ways of producing the toxic molecule specifically in the seed can be envisaged. In all procedures, at least one step in the production of the cytotoxic molecule has to occur specifically within the seed, the endosperm or the tegument. For example, a lamS gene (gene 1 for auxin) constitutively expressed in a plant can be used, and subsequently cross that plant with a plant containing the IamH gene (gene 2 for auxin) under the control of a seed-specific promoter. , so that AMI is produced in all cells of the plant, but the growth regulator IAA is produced only in seed cells due to the action of the specific lamH gene thereof. Conversely, it is possible to have lamH constitutively expressed in a plant, and to cross this plant with a plant containing a specific seed promoter directing the lamS gene. In this situation, the growth regulator IAA is only produced in seed cells. In various embodiments, both the lamH gene and the lamS gene are placed under the control of seed-specific promoters, and preferably using the same seed-specific promoter or a similar promoter whose expression substantially overlaps with that of another promoter to direct independently the expression of these two genes. Additionally, by linking the lamH gene to a selectable agent such as a herbicide, the production of hybrid plants is greatly facilitated. Any number of genes can be used to carry out the method and methods of the invention, provided that the simultaneous production of one or more enzymatic or synthetic activities specifically in the seed leads to the production of a substance that is toxic or inhibitory of the normal production of the seed, or interfere specifically with the seed coat or the development of the endosperm. This implies that one or more of these activities may be constitutive in the plant, but that the final combination of all enzymatic activities be limited to the seed. As can be seen, various combinations of cytotoxic genes and promoters specific to seed development 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 grown in plants that produce only seedless fruits. This system is economically favorable, because for each cross-pollination, a large number of seedless fruits are 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 to develop a hybrid crop, including the most valuable combination of hybrid features and vigor. This is achieved in the same manner as described above, except that the cytotoxic gene is expressed from a tegument specific promoter. The tegument is the excrescence of the integuments, a strictly maternal tissue. Therefore, the hybrid crossing carried by gene 1 for interruption together with gene 2 does not involve this tissue of the tegument. The seed coat Fl has gene 1 or gene 2, depending on which one is used as the female parent, and thus the seed Fl develops normally. Then, the seed Fl gives rise to a fruit carrier plant Fl, and all the vegetative cells (including the cells of the tegument) inherit either the gene 1 co or the gene 2 of the embryo. Thus, the tegument of the plant Fl has a gene for activated cell disruption. The tegument is an essential tissue for the development and viability of the seed. When the seed is fully mature, the tegument serves as a protective layer of the internal parts of the seed. During the development of the seed, the tegument is a vital tissue for nutrient uptake for the developing embryo. The seed is nutritionally "parasitic" for the mother plant. All the raw materials necessary for the growth of the seed must be acquired. In the seeds of dicotyledonous plants, the vascular tissue enters the seed through the funiculus and then anastomoses in the tissue of the tegument. There is no connection of vascular tissue or union by plasmodesms between the tegument and the embryo. Therefore, all nutrient solutes released in the developing seed must be released into the tegument and then translocated by diffusion to the embryo. Techniques have been developed to study the composition of nutrients in the integument [Hsu et al., Plant Physiol. 75: 181 (1984); Thorne & Rainbird, Plant Physiol. 72: 268 (1983); Patrick, J. Plant Physiol. 115: 297 (1984); Wolswinkel & Ammerlaan, J Exp. Bot. 36: 359 (1985)], and also the detailed cellular mechanisms of solute release (Offler &Patrick, Aust., J. Plant Physiol 11:79 (1984); Patrick, Physiol. Plant, 78: 298 (1990) It is obvious that the destruction of this vital nutrient-concentrating tissue causes abortion of the seed.

Several vectors were constructed according to the methods of the invention: Gene 1-pal 1425 (promoter Bp 4 f gene 1) Gene 2-pal 1426 (promoter Bp 4 + gene 2) The vectors were transformed according to Moloney and others, 1989, Pl. Cell Reports 8: 238-242. Seed was obtained from each event, as well as crossings were made with each event. After the plants bloomed and tried to produce seeds, it was observed that the pods were empty in plants with gene 1 and gene 2, as well as in plants with only gene 1.

EXAMPLE 2 Several vectors have been constructed in accordance with the invention, and include: Supermass: PAT: PIN:: DC3: GEN 1-GEN 2: IN Super ass: PAT: PIN :: Napina: GEN 1 - GEN 2: PIN Supermass: PAT: PIN: Faseolin: GEN 1- GEN 2: PIN The marker selectable for these vectors is the Supermass promoter operably linked to the PAT gene that conditions resistance to bialaphos (a herbicide), and the PIN II terminator (a polyadenylation signal). The three promoters for GEN 1 - GEN 2 are all seed specific and should result in toxicity for the development of the same. The degree of inhibition of seed development will depend on when and where the promoters work. The primary transgenic events must show segregation in the seed, and an expected phenotype is the 3: 1 ratio of non-full seed: normal seed.

EXAMPLE 3 A primary objective is to demonstrate in principle that the gene 1: gene 2 under the control of a seed-specific promoter, is capable of stopping the development of the same without adversely affecting the development of the fruit, that is, the genes function in a tissue-specific and autonomous way of the cell. Melon is the alternative for plant species due to its short life cycle and relatively small greenhouse space requirements.

Expression vectors will be used in plants in which both gene 1 and gene 2 are placed under the control of the DC3, napine or phaseolin promoters, to test the specific temporal and tissue character, i.e., early development of the seed in the melon. An expected phenotype would be transgenic TO that produce fruits that have 75% of seeds aborted when they are self-fertilized, or 50% of seeds aborted when they cross with a wild-type plant. Control plants that have been regenerated from tissue culture for percentage of aborted seeds will be compared to ensure that the phenotype is not the result of other factors. The molecular analysis of the individual seeds will also confirm the presence of transgenes, and of cosegregation of normal seeds against aborted seeds. In addition to the functional evaluation of the toxicity of gene l: gene 2, another evaluation of the promoter can be made with the cytochemical GUS marker and several seed-specific promoters. It is likely that it will be necessary to modify the existing promoters, or that it is necessary to isolate other promoters (see below) that have temporal and tissue expression compatible with the needs of this application.

EXAMPLE 4 Evaluation of gene l: gene 2 for hybrid seed production systems Two systems will be evaluated for hybrid seed production efficiency. The first is the use of a specific promoter of the embryo: gene such as DC3 :: Gen 1 in the male parent, and DC3 :: Gen 2 in the female parent. Both parents must produce seed normally when they are self-fertilized or improved for seed production. However, an abnormal floral phenotype has been observed with some combinations of anther-specific promoters and Gen 1 in Brassica (Amoldo, 1995, August monthly report, Cañóla Transformation). The degree of phenotypic abnormality will depend on the specific promoter, although the suggested promoters should have much less phenotypic disturbance than those under the control of constitutive promoters (Sitbon et al., 1992, "Transgenic Tobacco Plants Coexpressing the Agrobacterium Tumefaciens IAAM and IAAH Display genes Altered Growth and Indoleacetic Acid Metabolism ", Plant Physiol. 99: 1062-1069; Klee et al., 1987, "The Effects of Overproduction of Two Agrobacterium Tumefaciens T-DNA Auxin Biosynthetic Gene Products in Transgenic Petunia Plants ", Genes Dev. 1: 86-96).

Phenotypic and molecular analyzes of seed production in the component parental combinations should verify the nature of seed maturation in both parent combinations. One possible difficulty with this hybrid seed production alternative is that both gene 1 and gene 2 will be potentially expressed in the female parent used for seed production. The embryo is diploid and seed specific, and if the expression of the component genes is made from equal contributions of male and female gametes, the female parent in the seed production block will be seedless. An alternative to this procedure would be to release a hybrid that is homozygous for gene 2 and that is male sterile or gynoic. A pollen donor containing gene 1 that would pollinate the androsterile hybrid containing gene 2 would also be provided. All the fruits obtained from the hybrid would be "seedless". Another alternative for this method would be to repress the transcription activity of gene 1 in the female parent through the use of an inducible promoter. The Lex A gene (Brent and Ptashne, 1985, "A Eukaryotic Trans-Activator Activator Bearing the DNA Specificity of a Prokaryotic Repressor", Cell, 43: 729-736) is said useful promoter. Gene 1 would normally be expressed in the female parent, except during seed production of the hybrid in which the "inducer" is used to repress the gene. Thus, seed production would be "normal" as long as the Fl hybrid sold to farmers is seedless. An alternative compatible with the conventional production of hybrid seed, is to put both gene 1 and gene 2 under the control of a specific tegument promoter. Because the tegument is of maternal origin, the female parent producing hybrid seed would not express the seedless phenotype when it crosses a male parent that contains gene 2, since the maternal tissue would only express gene 1. The genetic constitution of the embryo Fl contains both gene 1 and gene 2, and in the farmer field they would be expressed in the maternal tissue and have a seedless phenotype. The differential deployment technique can be used using Rt-PCR to take advantage of selectively isolating the message produced in the tissue of the tegument, comparatively with specific tissue of the embryo and specific tissue of the fruit. This technique has been used in several applications, including the isolation of cancer genes and the isolation of genes related to fruit ripening in tomatoes (Oh et al., 1995, "A Modified Procedure for PCR-Based Differential Display and Demonstration of use in Plants for Isolation of Genes Related to Fruit Ripening, "Plant Mol. Rpt., 13: 70-81.

Claims (27)

NOVELTY OF THE INVENTION CLAIMS

1. - An expression construct capable of producing transgenic plants that will produce fruits with seeds of a diminished size or a very small number of seeds, characterized in that it comprises: a recombinant gene or combination of genes that encode the expression of a cytotoxic protein; and a seed-specific promoter operably linked to said gene or genes.

2. The expression construct according to claim 1, characterized in that said combination of genes includes: a first DNA sequence that codes for a first genetic product that is capable of making a non-toxic substance cytotoxic to a cell of a plant; and a second DNA sequence encoding a second genetic product that is a non-toxic substance or that encodes a second genetic product that is capable of converting an endogenous substance for a plant cell into a non-toxic substance, said non-toxic substance being made cytotoxic by said first genetic product.

3. The expression construct according to claim 2, characterized in that said first DNA sequence encodes indole acetamide hydrolase (lamH), and wherein said second DNA sequence encodes indole acetamide synthetase (lamS).

4. The expression construct according to claim 1, characterized in that said cytotoxic gene encodes DAM methylase.

5. The expression construct according to claim 1, characterized in that said promoter is a maternal tissue promoter.

6. The expression construct according to claim 1, characterized in that said promoter is selected from the group consisting of: the napin promoter, the phaseolin promoter and the DC3 promoter.

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

8. An expression construction for the production of a transgenic parental line that when crossed with a second parental line that contains a DNA sequence that codes for a second genetic product that is a non-toxic substance or that codes for a second product genetic that is capable of converting an endogenous substance for a plant cell into a non-toxic substance, said non-toxic substance being made cytotoxic by said first genetic product, will produce a plant Fl that will develop fruits with seeds of a diminished size or a very small number of seeds, characterized in that it comprises: a DNA sequence coding for a first genetic product that is capable of causing a non-toxic substance to be cytotoxic to a cell of a plant, said first genetic product being operably linked to a specific promoter of the seed.

9. An expression construct for the production of a transgenic parental line that, when crossed with a second parental line containing a DNA sequence regulated by a seed-specific promoter that codes for a first genetic product that is capable of causing a non-toxic substance to be cytotoxic to a cell of a plant, will produce a plant Fl that will develop fruits with seeds of a diminished size or a very small number of seeds, characterized in that it comprises: a DNA sequence that codes for a second product genetic that is a non-toxic substance or that codes for a second genetic product that is capable of converting an endogenous substance for a plant cell into a non-toxic substance, said non-toxic substance being made cytotoxic by said first genetic product.

10. A nucleic acid vector, characterized in that it comprises the expression constructions according to claims 1, 8 or 9.

11. The vector according to claim 10, characterized in that said vector is a cloning vector.

12. - The vector according to claim 10, characterized in that said vector is an expression vector.

13. The vector according to claim 10, characterized in that it also comprises a marker gene for selection of transformed cells. 14.- The vector in accordance with the claim 12, characterized in that said marker gene is selected from the group consisting of a gene for resistance to ampicillin, a gene for resistance to tetracycline and a gene for resistance to hygromycin. 15. The vector according to claim 10, characterized in that it comprises a polyadenylation signal. 16. A prokaryotic or eukaryotic host cell, characterized in that it is transformed with the nucleic acid vector according to claim 10. 17.- A transgenic plant, characterized in that it comprises a plant cell or ancestor thereof that have been transformed with the vector according to claim 10. 18. A method for producing a hybrid seed that will produce a plant that, when pollinated, will produce fruits with seeds of a diminished size, or a very small number of seeds, characterized in that it comprises : pollinate a parent plant that has been transformed, or whose ancestor has been transformed with a first DNA sequence that codes for a first genetic product that is capable of making a non-toxic substance cytotoxic to a cell of a plant, said sequence of DNA being operably linked to a specific promoter of maternal tissue; and with a second parent plant that has been transformed, or whose ancestor has been transformed with a second DNA sequence that codes for a second genetic product that is a non-toxic substance, or that codes for a second genetic product that is capable of converting an endogenous substance for a plant cell in a non-toxic substance, said DNA sequence being operably linked to a specific promoter of maternal tissue, and said non-toxic substance that has been made cytotoxic by said first genetic product. 19.- The method according to the claim 18, characterized in that said maternal tissue-specific promoter is a tegument-specific promoter. 20.- A hybrid seed, characterized in that it is produced by the method according to claim 18. 21.- A fruit with seeds of diminished size or very few seeds produced by a plant that contains a DNA sequence that codes for a protein cytotoxic expressed during the development of the seed. 22. A method for producing fruits with seed of diminished size or very few seeds, characterized in that it comprises: transforming an angiosperm plant cell with a DNA sequence coding for a cytotoxic gene operably linked to a specific promoter of the seed; generate a plant from said transformed cell; and allowing said plant to be pollinated, so that the development of the seed is initiated and then stopped without interrupting the formation of the fruit. 23. The method according to claim 22, characterized in that said plant is a melon plant. 24.- The method of compliance with the claim 22, characterized in that said DNA sequence codes for indole acetamide hydrolase (lamH) and indole acetamide synthetase (lamS). 25. The method according to claim 22, characterized in that said promoter is selected from the group consisting of: the napin promoter, the phaseolin promoter and the DC3 promoter. 26.- A method to produce a hybrid plant that, when pollinated, will produce fruits with seeds of a diminished size, or a very small number of seeds, characterized because it includes: pollinating a progenitor plant that has been transformed, or whose ancestor has been transformed with a first DNA sequence encoding a first genetic product that is capable of making a non-toxic substance cytotoxic to a cell of a plant, said DNA sequence being operably linked to a seed-specific promoter; and with a second parent plant that has been transformed, or whose ancestor has been transformed with a second DNA sequence that codes for a second genetic product that is a non-toxic substance, or that codes for a second genetic product that is capable of converting an endogenous substance for a plant cell in a non-toxic substance, said DNA sequence being operably linked to a seed-specific promoter, and said non-toxic substance that has been made cytotoxic by said first genetic product. 27. The expression construct according to claim 26, characterized in that said first DNA sequence encodes indole acetamide hydrolase (lamH), and wherein said second DNA sequence encodes indole acetamide synthetase (lamS).