WO1997030165A2 - Method for the induction of genetic parthenocarpy in plants - Google Patents

Method for the induction of genetic parthenocarpy in plants Download PDF

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WO1997030165A2
WO1997030165A2 PCT/IL1997/000051 IL9700051W WO9730165A2 WO 1997030165 A2 WO1997030165 A2 WO 1997030165A2 IL 9700051 W IL9700051 W IL 9700051W WO 9730165 A2 WO9730165 A2 WO 9730165A2
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plant
plants
fruit
plasmid
ovary
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PCT/IL1997/000051
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French (fr)
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WO1997030165A3 (en
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Rivka Barg
Yehiam Salts
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State Of Israel/Ministry Of Agriculture
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Priority to US09/125,287 priority patent/US6114602A/en
Priority to EP97902555A priority patent/EP1007704A2/en
Publication of WO1997030165A2 publication Critical patent/WO1997030165A2/en
Publication of WO1997030165A3 publication Critical patent/WO1997030165A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8233—Female-specific, e.g. pistil, ovule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8291—Hormone-influenced development
    • C12N15/8294—Auxins

Definitions

  • the present invention concerns a method for production of parthenocarpic plants and more particularly it is a method for producing genetic parthenocarpy.
  • Parthenocarpy may either be artificially induced in normal plants or may occur naturally as the result of a genetic trait. Genetic parthenocarpy may be obligatory or facultative. In the former seeds are never formed. In facultative parthenocarpy seedless fruit is produced when environmental conditions are unfavorable for pollination and fertilization. However, when fertilization does occur seeded fruits are formed, which enables reproductive propagation of the facultative parthenocarpic plants.
  • Parthenocarpy is discussed in various scientific articles, such as:
  • Agrobacterium rhizogenes inserts T-DNA into the genome of host plant root cells.
  • Plant tissue culture manual (Ed. K. Lindsey) B6:1-9.
  • Agrobacterium rhizogenes involves the transfer of plasmid DNA to the plant genome.
  • auxins In the tomato, the most effective treatments for induction of parthenocarpy utilize auxins, synthetic auxins or auxin transport inhibitors (reviewed by Ho and Hewitt 1986, Varga and Bruinsma 1986, Abad and Monteiro 1989).
  • this treatment is very laborious since the auxin has to be applied to each truss separately, to avoid the adverse effects characteristic to auxin application to the whole plant.
  • auxin also appears to play a role in genetic (natural) parthenocarpic fruit set.
  • Gustafson (1939a,b) found that auxin concentrations in ovaries of parthenocarpic orange, lemon and grape varieties were significantly higher than in seeded varieties.
  • Nitsch (1970) suggested that natural parthenocarpy is related to the ability of seedless varieties to establish a threshold concentration of hormones required for fruit set at anthesis.
  • rolB gene which is included in TL-DNA of the Agrobacterium rhizogenes agropine-type Ri-Plasmid. (Chilton et al. 1982, Spano et al. 1982, White et al. 1982, Willmitzer et al. 1982, Peterson et al. 1989).
  • the present invention provides an improved method for the production of genetic parthenocarpy in plants.
  • a method for the production of genetic parthenocarpy in plants which include the steps of providing a cassette including a DNA sequence coding for modulation of auxin effects in plants, and a promoter specific for the ovary between anthesis and early fruit development to control the DNA sequence, introducing the cassette into a plant.
  • the method also includes the step of screening the plant for either facultative or obligatory parthenocarpic characteristics.
  • the step of introducing includes the steps of transformation of plant material, and regeneration of transformed plants.
  • the plant material includes seed derived cotyledons.
  • the step of transformation includes the steps of providing a plasmid incorporating the cassette, introducing the plasmid into A. tumerfaciens, and incorporating the plasmid into the plant material by co-cultivation with the A. tumerfaciens, including the plasmid.
  • the DNA sequence coding for modulation of auxin effects in plants includes the sequences of the rolB gene.
  • the step of screening includes growing the plant to produce fruit and examining the fruit for seed production under fertility permissive conditions. In accordance with still a further preferred embodiment of the present invention the step of screening includes growing the plant to produce fruit and examining the fruit for seed production under fertility restrictive conditions.
  • the method also includes the step of screening the plant for either facultative or obligatory parthenocarpic characteristics.
  • the tomato plant includes the fruit of the tomato plant.
  • Fig. 1 is a diagrammatic analysis of GUS expression in transgenic plants harboring various deletions of the TPRP-F1 promoter sequence;
  • the column a indicates the number of independent transformations.
  • Columns b describe data obtained from quantitative fluorometric assay with 4MU.(++ indicates high fluorescence, + intermediate high, +/- intermediate low, - no fluorescence).
  • Column c is based on histochemical assay with X - Glue. (++ indicates high color developed within 2 hr., + high color developed upon overnight incubation, +/- faint color developed upon overnight incubation, - no color developed upon overnight incubation).
  • Fig. 2 is a diagram of the construction of plasmid pSB171; The 7Kbp HindIII fragment of ⁇ G16 was inserted into the HindIII site of plasmid pBluescript SK+.
  • Fig. 3 is a diagram of the construction of pSBN28; The NsiI fragment of pBS171 was inserted into the Pst I site of pBluescript.
  • Fig. 4 is a diagram of the construction of plasmid pPSN33; The mutated pTPRP-Fl fragment was inserted into the Xba I and Kpn I sites of pBluescript KS+.
  • Fig. 5 is a diagram of the construction of plasmid pBSrolB; The EcoRI - HindIII fragment of pUC19-B26 was ligated into the corresponding sites of pBluescript
  • Fig. 6 is a diagram of the construction of plasmid pBSNrolB; The Kpn I fragment of pBSro1B was ligated into the Kpn I site of pBSN33.
  • Fig. 7 is a diagram of the construction of plasmid pGB18; The multiple cloning site of pGA492 was replaced by the one of pUC19.
  • Fig. 8 is a diagram of the construction of plasmid pGB18ro1B; The Xba-HindIII fragment of pBSNro1B was ligated into the corresponding sites of pGB18..
  • Table 1 shows the sequence of the TPRP-F1 genomic clone, including 2643 bp of the promoter region.
  • the sequence of the promoter region extending from 1-2483 was not published earlier.
  • the sequence extending from 2484-4320 was published earlier (Salts et al. 1992).
  • the ATG codon printed in bold letters is the TPRP-F1 translation initiation codon.
  • the underlined bold G is the TPRP-F1 genomic clone, including 2643 bp of the promoter region.
  • the sequence of the promoter region extending from 1-2483 was not published earlier.
  • the sequence extending from 2484-4320 was published earlier (Salts et al. 1992).
  • the ATG codon printed in bold letters is the TPRP-F1 translation initiation codon.
  • the underlined bold G is the TPRP-F1 translation initiation codon.
  • Table 2 shows the sequence of the TPRP-F1 promoter included in the binary plasmid pGB18ro1B (Fig.7). The sequence corresponds to 101-2643 in Table 1.
  • Table 3 shows a shorter promoter that confers ovary and embryo specificity (From
  • Table 4 shows an alternative combination of sequence from the TPRP-F1 promoter that confers ovary and developing embryo specificity (From 1-1728 fused to 2079- 2643, Table 1)
  • Table 5 shows the sequence of the rolB gene included in the binary vector pGB18ro1B ( see Fig.5). Trie sequence is that presented in Slightom et al 1986. The translation initiation codon (position 40) is presented in bold letters
  • Table 6 shows seed bearing phenotype of the various transgenic plants (R o ) and their progenies (R 1 )
  • the method for production includes providing a cassette including a DNA sequence coding for modulation of auxin effects, and a promoter specific for the ovary between anthesis and early fruit production to control the DNA sequence, introducing the cassette into a plant and screening the plant for parthenocarpic characteristics.
  • Bacterial strains and cultures The E. Coli strain DH5 ⁇ (Raleigh et al. 1989) served for all the plasmid construction steps, and strain SM10 (Simon et al. 1983) served for conjugation of the plasmids into Agrobacteruim tumefaciens. PCR, DNA and RNA analysis, and recombinant plasmids construction were performed according to established procedures: (Ausubel et al. 1987, Sambrook et al. 1989)
  • the promoter of the TPRP-F1 gene was isolated following the characterization of the TPRP-F1 gene as an ovary and early fruit specific one, and following sequencing of the gene's coding region (Salts et al.1991, 1992). Later on a genomic clone was isolated. The sequence of the geno mic clone including putative promoter region extending 2643 bp 5' to the translation initiation codon is presented in Table 1. The sequenced of the coding region, including 20 bp 5' to the translation initiation codon (position 2623 in Table 1) was published previously (Salts et al.1992), while the sequence extending between 1-2622 (Table 1) was not published.
  • a preliminary functional analysis of the promoter was performed by fusion of various deletions of the putative promoter sequence to the reporter gene uidA (GUS), and analysis of GUS expression (Jefferson et al.1987) in stable transgenic tomato plants. The results of the preliminary analysis were briefly reported (Carmi et al. 1994). Primer extension analysis indicated that the transcribed transcript of the TPRP-F1 gene includes 408 bp of untranslated leader sequence. A detailed analysis of the promoter region of the TPRP-F1 gene was performed including additional deletions and testing GUS expression in more organs and at additional developmental stages. The results of this analysis are summarized in Figure 1.
  • the recombinant plasmid pSB171 (Fig. 2) was obtained by subcloning the genomic
  • PCR product was digested with KpnI and XbaI and ligated to pBluescript creating the plasmid pBSN33 (Fig. 4).
  • pUC19-B26 harbors the sequence between nucleotides 9814 and 11324 according to Slighton et al. (1986).
  • the sequence of rolB included in this plasmid is given in Table 5.
  • the EcoRI - HindIII fragment of this plasmid was isolated and cloned into the EcoRI/HindIII-restriction cleavage sites of the plasmid pBluescript to form pBSrolB (Fig. 5).
  • the KpnI fragment of pBSrolB was isolated and subcloned into the KpnI site of pBSN33, and a clone with the rolB gene inserted in the correct orientation was identified by restriction enzyme analysis.
  • This plasmid was named pBSNrolB (Fig. 6).
  • the EcoRI-HindIII polylinker of plasmid pUC18 was isolated and subcloned into the EcoRI/HindIII-restriction cleavage sites of the plasmid pGA492 (An 1986) to form plasmid pGB18 (Fig. 7).
  • the XbaI-BamHI fragment of pBSNrolB was isolated and subcloned into the XbaI/BamHI restriction cleavage sites of plasmid pGB18 to form plasmid pGB18rolB (Fig. 8).
  • the binary vector pGB18rolB was transformed into the indeterminate tomato breeding line MP-1 by co-cultivation of cotyledons derived from 10-day-old seedlings with Agrobacterium tumefaciens strain EAH105 (Hood et al. 1993). Essentially, the protocol described by McCormick (1991) was followed, except that the 'overnight' bacteria culture was resuspended in liquid MS (Murashige and Skoog 1962) medium before pouring over the cotyledons, and using 0.5 mg/L cefotaxime instead of carbenicillin. The cotyledons and the plantlets regenerated from them were propagated in the presence of 100 mg/L of kanamycin until rooted. Regenerated plants were planted in peat pellets (Jiffy-7, a/s Jiffy Products, Norway) for hardening (for 1-2 weeks) and then transferred to insect-proof greenhouse or net-house.
  • transgenic plants were germinated on selective medium (1/2 MS medium, 3% sucrose and 100 mg/L Kanamycin), where only transgenic seedlings develop a branched root system. Description of the transgenic plants
  • Ro Three parthenocarpic primary (Ro) transgenic plants were regenerated from breeding line MP-1: MPB-4, MPB-12 and MPB-13, and MPB-19 . Transformation of the Ro plants was confirmed by PCR analysis, using primers for the selectable marker gene nptII, and by Southern analysis, using a KpnI fragment of the rolB gene as a probe.
  • Plants MPB-12 and MPB-13 behaved as facultative parthenocarpic ones. Their seedless fruits were full of jelly and contained 4 locules or more. In most MPB-13 fruits and in some of the MPB-12 fruits the columella was somewhat greater than in the parental line fruits. In some of the MPB-13 fruits the jelly was of greenish hue, especially in fruits developed at high temperatures. This phenomenon was also reported for auxin-induced fruits.(Abad & Monteiro 1989). The fasciation typical to auxin-induced parthenocarpic fruits was not observed in the seedless fruits of MPB-12 and MPB-13.
  • Vegetative growth habit of R1 plants Most of the Kan r seedlings of PMB-12-R 1 and all of the Kan r MPB-13-R 1 plants were characterized by broad cotyledons which rolled downwards. The severity of the phenomenon varied among the various plants. The root system of some of the MPB-13-R 1 seedlings was more developed than that of the control plants. The developed plants did not manifest auxin related malformations (such as leaf epinasty, stem curling or adventitious root development.
  • Seedlessness and fruit characteristics in R1 plants As specified in Table 6, most of the fruits developed on the various Kan r MPB-12-R1 plants were seedless and full of jelly. Occasionally seeded fruits developed, most of them contained significantly less seeds than the parental cultivar, and the average weight and size of the seeds was significantly higher than that of the MP-1 fruits. The frequency of seeded fruits varied among the various R 1 plants. The fruits were not puffy; some of the fruits were of slightly flattened oval shape. Transverse constriction was not observed, apparently because the fruits did not develop under exceptionally high temperatures. Most of the fruits of the various MPB-13-R 1 plants were of regular shape, seedless, and full of jelly. Few seeded fruits developed, and most of them contained less seeds than MP-1.
  • Vegetative growth habit of R2 plants The phenomenon of broad cotyledons was also observed in the kan r R 2 seedlings of MPB-12 and MPB-13. The developing plants were of normal growth habit.

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Abstract

The present invention provides an improved method for the production of genetic parthenocarpy in plants, which includes the steps of: providing a cassette including a DNA sequence coding for modulation of auxin effects in plants, such as sequences of the rolB gene, and a promoter specific for the ovary between anthesis and early fruit development to control the DNA sequence and introducing the cassette into a plant. Preferably, the DNA is introduced by transformation of plant material (including seed derived cotyledons), and regeneration of transformed plants. Preferably the method also includes the step of screening the plant for either facultative or obligatory parthenocarpic characteristics.

Description

METHOD FOR THE INDUCTION OF GENETIC PARTHENOCARPY IN PLANTS
FIELD OF THE INVENTION
The present invention concerns a method for production of parthenocarpic plants and more particularly it is a method for producing genetic parthenocarpy.
BACKGROUND OF THE INVENTION
Fruit setting and development normally depend on successful fertilization. In tomato and many other species, a major limiting factor for fruit setting is the extreme sensitivity of the pollen production to moderately high or low temperatures and inadequate humidity (Picken 1984). Parthenocarpy, which is the ability to set seedless fruits, enables to circumvent these environmental constraints on fruit production. Consequently, in many fruit bearing plants parthenocarpy is of considerable economic significance.
Parthenocarpy may either be artificially induced in normal plants or may occur naturally as the result of a genetic trait. Genetic parthenocarpy may be obligatory or facultative. In the former seeds are never formed. In facultative parthenocarpy seedless fruit is produced when environmental conditions are unfavorable for pollination and fertilization. However, when fertilization does occur seeded fruits are formed, which enables reproductive propagation of the facultative parthenocarpic plants.
Parthenocarpy is discussed in various scientific articles, such as:
Abad M, Monteiro AA (1989) The use of auxins for the production of greenhouse tomatoes in mild-winter conditions: A review. Sci Hort 38: 167-192.
An G (1986) Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells. Plant Physiol 81:86-91.
Ausubel FM, Kingston RE, Moore DD, Smith JA, Seidman JG, Struhl K (1988)
Current protocols in molecular biology. Wiley InterScience. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Amer J Bot 50:859-865.
Carmi N, Barg R, Salts Y (1994). Expression of a tomato young fruit specific proline-rich coding gene. In Abstracts of 4th International Congress of Plant Molecular
Biology. Amsterdam (Abs. 538).
Chilton MD, Tepfer DA, Petit A, David C, Casse-Delbart F, Tempe J (1982)
Agrobacterium rhizogenes inserts T-DNA into the genome of host plant root cells.
Nature 295:432-434.
Gustafson FG (1939a) The cause of natural parthenocarpy. Amer J Bot 26: 135-138.
Gustafson FG (1939b) Auxin distribution in fruits and its significance in fruit development Amer J Bot 26: 189-194.
Ho LC, Hewitt JD (1986) Fruit development. In: The Tomato Crop. A Scientific
Basis for Improvement (Athernon JR and Rudich J, Eds), Chapman and Hall,
London, New York Pp. 201-239.
Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993). New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. 2: 208-218.
efferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901-3907.
McCormick S (1991) Transformation of tomato with Agrobacterium tumefaciens. In:
Plant tissue culture manual (Ed. K. Lindsey) B6:1-9.
Murashige T, Skoog F A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant (1962)
Nitsch JP (1970) Hormonal factors in growth and development In: The biochemistry of fruits and their products (AC Hulme ed). Vol 2, Academic Press,
London . Pp.428-47
Peterson SG, Stummann BM, Olesen P, Henningsen KW (1989) Structure and function of root-inducing (Ri) plasmids and their relation to tumor-inducing (Ti) plasmids. Physiol Plant 77:427-435.
Picken AJ (1984) A review of pollination and fruit set in the tomato (Lycopersicon esculentum Mill .) J Hort Sci 59: 1-13. Raleigh EA, Lech K, Brent R (1989) In Current protocols in molecular biology (Eds.
Ausubel FM et al. Publishing associates and Wiley INterscience NY, Unit 1.4
Salts Y , Wachs R, Gruissem W, Barg R (1991) Sequence coding for a novel proline-rich protein preferentially expressed in young tomato fruit. Plant Mol Biol 17:
149-150.
Salts Y, Kenigbuch D, Wachs R, Gruissem W, Barg R (1992) DNA sequence of the tomato fruit expressed proline-rich protein gene TPRP-F1 reveals an intron within the
3'-untranslated transcript Plant Mol Biol 18:407-409.
Sambrook J, Fritsch EF, Maniatis T (1989). Molecular Cloning. A laboratory manual.
Second edition. Cold Spring Harbor Laboratory Press, USA.
Schmulling T, Schell J, Spena A (1988) Single genes from_Agrobacterium rhizogenes influence plant development. EMBO J 7: 2621-2629.
Simon R, Preifer U, Puhler A (1983) A broad host mobilization system for in vitro genetic engineering: transposon mutagenesis in Gram-negative bacteria.
Bio/technology 1:784-791.
Slightom JL, Durand-Tardif M, Jouanin L, Tepfer D (1986) Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes Agropine type plasmid. Identification of open reading frames. J Biol Chem 261:108-121.
Spano L, Pompom M, Costantino P, van Slogteren GMS and Tempe J (1982) Identification of T-DNA in the root-inducing plasmid of the agropine type Agrobacterium rhizogenes 1855. Plant Mol Biol 1: 291-300.
van Altvorst AC, Bino RJ, van Dijk AJ, Lamers AMJ, Lindhout WH, van der Mark F, Dons JJM (1992) Effects of the introduction of Agrobacterium rhizogenes rol genes on tomato plant and flower development. Plant Sci 83:77-85.
Varga A, Bruinsma J (1986) Tomato. In: CRC Handbook of Fruit Set and Development (Monselise SP, ed .) CRC Press Inc. Boca Raton, FL. Pp. 461-481.
White FF, Ghidossi G, Gordon MP, Nester EW (1982) Tumor induction by Agrobacterium rhizogenes involves the transfer of plasmid DNA to the plant genome. Proc Natl Acad Sci USA 79: 3193-3197.
Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Thechnol 17: 189-194. Picken AJ (1984) A review of pollination and fruit set in the tomato (Lycopersicon esculentum Mill .) J Hort Sci 59: 1-13.
Raleigh EA, Lech K, Brent R (1989) In Current protocols in molecular biology (Eds.
Ausubel FM et al. Publishing associates and Wiley INterscience NY, Unit 1.4
Salts Y , Wachs R, Gruissem W, Barg R (1991) Sequence coding for a novel proline-rich protein preferentially expressed in young tomato fruit. Plant Mol Biol 17:
149-150.
Salts Y, Kenigbuch D, Wachs R, Gruissem W, Barg R (1992) DNA sequence of the tomato fruit expressed proline-rich protein gene TPRP-F1 reveals an intron within the
3'-untranslated transcript. Plant Mol Biol 18:407-409.
Sambrook J, Fritsch EF, Maniatis T (1989). Molecular Cloning. A laboratory manual.
Second edition. Cold Spring Harbor Laboratory Press, USA.
Schmulling T, Schell J, Spena A (1988) Single genes from_Agrobacterium rhizogenes influence plant development. EMBO J 7: 2621-2629.
Simon R, Preifer U, Puhler A (1983) A broad host mobilization system for in vitro genetic engineering: transposon mutagenesis in Gram-negative bacteria.
Bio/technology 1:784-791.
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In the tomato, the most effective treatments for induction of parthenocarpy utilize auxins, synthetic auxins or auxin transport inhibitors (reviewed by Ho and Hewitt 1986, Varga and Bruinsma 1986, Abad and Monteiro 1989). However, this treatment is very laborious since the auxin has to be applied to each truss separately, to avoid the adverse effects characteristic to auxin application to the whole plant.
Auxin also appears to play a role in genetic (natural) parthenocarpic fruit set. For example, Gustafson (1939a,b) found that auxin concentrations in ovaries of parthenocarpic orange, lemon and grape varieties were significantly higher than in seeded varieties. Based on similar data, Nitsch (1970) suggested that natural parthenocarpy is related to the ability of seedless varieties to establish a threshold concentration of hormones required for fruit set at anthesis.
Several genes which control the effect of auxin in plants are known in the art. One such gene, for example, is the rolB gene which is included in TL-DNA of the Agrobacterium rhizogenes agropine-type Ri-Plasmid. (Chilton et al. 1982, Spano et al. 1982, White et al. 1982, Willmitzer et al. 1982, Peterson et al. 1989).
Transgenic plants expressing the rolB gene alone manifest several syndromes characteristics of auxin toxicity, such as leaf abnormalities, increased stigma and flower size, heterostyly and increased formation of adventitious roots on the stem (Schmulling et al. 1988). van Altvorst et al. (1992) described the phenotypic effects of the various rol genes in transgenic tomato, however, they did not report parthenocarpic fruit development when the rolB gene was inserted. SUMMARY OF THE INVENTION
The present invention provides an improved method for the production of genetic parthenocarpy in plants.
There is thus provided in accordance with a preferred embodiment of the present invention a method for the production of genetic parthenocarpy in plants which include the steps of providing a cassette including a DNA sequence coding for modulation of auxin effects in plants, and a promoter specific for the ovary between anthesis and early fruit development to control the DNA sequence, introducing the cassette into a plant.
Preferably the method also includes the step of screening the plant for either facultative or obligatory parthenocarpic characteristics.
In accordance with a preferred embodiment of the present invention the step of introducing includes the steps of transformation of plant material, and regeneration of transformed plants.
In accordance with another preferred embodiment of the present invention the plant material includes seed derived cotyledons.
In accordance with yet another preferred embodiment of the present invention the step of transformation includes the steps of providing a plasmid incorporating the cassette, introducing the plasmid into A. tumerfaciens, and incorporating the plasmid into the plant material by co-cultivation with the A. tumerfaciens, including the plasmid.
In accordance with still another preferred embodiment of the present invention the DNA sequence coding for modulation of auxin effects in plants, includes the sequences of the rolB gene.
In accordance with a further preferred embodiment of the present invention the step of screening includes growing the plant to produce fruit and examining the fruit for seed production under fertility permissive conditions. In accordance with still a further preferred embodiment of the present invention the step of screening includes growing the plant to produce fruit and examining the fruit for seed production under fertility restrictive conditions.
Preferably the method also includes the step of screening the plant for either facultative or obligatory parthenocarpic characteristics.
In accordance with a preferred embodiment of the present invention the tomato plant includes the fruit of the tomato plant.
DESCRIPTION OF THE DRAWINGS
The present invention will be understood and appreciated more fully from the following detailed description taken in conjugation with the drawings and tables in which:
Fig. 1 is a diagrammatic analysis of GUS expression in transgenic plants harboring various deletions of the TPRP-F1 promoter sequence; In Fig. 1 the column a indicates the number of independent transformations. Columns b describe data obtained from quantitative fluorometric assay with 4MU.(++ indicates high fluorescence, + intermediate high, +/- intermediate low, - no fluorescence). Column c is based on histochemical assay with X - Glue. (++ indicates high color developed within 2 hr., + high color developed upon overnight incubation, +/- faint color developed upon overnight incubation, - no color developed upon overnight incubation). In row d the minimal -46 CaMV-35S promoter is installed in front of the GUS - ORF (open reading frame). In all other plasmids the 5'UTR (untranslated region of the TPRP-F1 promoter is retained.
Fig. 2 is a diagram of the construction of plasmid pSB171; The 7Kbp HindIII fragment of λG16 was inserted into the HindIII site of plasmid pBluescript SK+. Fig. 3 is a diagram of the construction of pSBN28; The NsiI fragment of pBS171 was inserted into the Pst I site of pBluescript.
Fig. 4 is a diagram of the construction of plasmid pPSN33; The mutated pTPRP-Fl fragment was inserted into the Xba I and Kpn I sites of pBluescript KS+. Fig. 5 is a diagram of the construction of plasmid pBSrolB; The EcoRI - HindIII fragment of pUC19-B26 was ligated into the corresponding sites of pBluescript
KS+.
Fig. 6 is a diagram of the construction of plasmid pBSNrolB; The Kpn I fragment of pBSro1B was ligated into the Kpn I site of pBSN33.
Fig. 7 is a diagram of the construction of plasmid pGB18; The multiple cloning site of pGA492 was replaced by the one of pUC19.
Fig. 8 is a diagram of the construction of plasmid pGB18ro1B; The Xba-HindIII fragment of pBSNro1B was ligated into the corresponding sites of pGB18..
Table 1 shows the sequence of the TPRP-F1 genomic clone, including 2643 bp of the promoter region. The sequence of the promoter region extending from 1-2483 was not published earlier. The sequence extending from 2484-4320 was published earlier (Salts et al. 1992). The ATG codon printed in bold letters (position 2644- 2647) is the TPRP-F1 translation initiation codon. The underlined bold G (position
2235) is the transcription initiation site (unpublished data).
Table 2: shows the sequence of the TPRP-F1 promoter included in the binary plasmid pGB18ro1B (Fig.7). The sequence corresponds to 101-2643 in Table 1.
Table 3: shows a shorter promoter that confers ovary and embryo specificity (From
943-2643 in Table 1)
Table 4: shows an alternative combination of sequence from the TPRP-F1 promoter that confers ovary and developing embryo specificity (From 1-1728 fused to 2079- 2643, Table 1)
Table 5: shows the sequence of the rolB gene included in the binary vector pGB18ro1B ( see Fig.5). Trie sequence is that presented in Slightom et al 1986. The translation initiation codon (position 40) is presented in bold letters
Table 6: shows seed bearing phenotype of the various transgenic plants (Ro) and their progenies (R1)
Table 7 : shows the analysis of characteristics of fruits developed on R2 transgenic plants and the parental line MP-1. Within columns values followed by common letters do not differ significantly at P=0.05. In table 7: 1) nP1= number of plants sampled, nFr= total number of fruits sampled from the given number of plants, fruits were collected between 14-30 June 1995.
2) The total number of fruits on the plant when sampled (only fruits greater than 15 mm were counted).
3) F and P values for the data presented in the column, Anova test was performed using Graphpad Instat 2.01 program for Macintosh, including Turkey test for analysis of all pairs within the column.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Reference is now made to the method involved in the production of genetic parthenocarpy in plants. The method for production includes providing a cassette including a DNA sequence coding for modulation of auxin effects, and a promoter specific for the ovary between anthesis and early fruit production to control the DNA sequence, introducing the cassette into a plant and screening the plant for parthenocarpic characteristics.
Materials & Methods
Bacterial strains and cultures: The E. Coli strain DH5α (Raleigh et al. 1989) served for all the plasmid construction steps, and strain SM10 (Simon et al. 1983) served for conjugation of the plasmids into Agrobacteruim tumefaciens. PCR, DNA and RNA analysis, and recombinant plasmids construction were performed according to established procedures: (Ausubel et al. 1987, Sambrook et al. 1989)
Example 1
Detailed analysis of the ovary and early fruit specific TPRP-F1 promoter
The promoter of the TPRP-F1 gene was isolated following the characterization of the TPRP-F1 gene as an ovary and early fruit specific one, and following sequencing of the gene's coding region (Salts et al.1991, 1992). Later on a genomic clone was isolated. The sequence of the geno mic clone including putative promoter region extending 2643 bp 5' to the translation initiation codon is presented in Table 1. The sequenced of the coding region, including 20 bp 5' to the translation initiation codon (position 2623 in Table 1) was published previously (Salts et al.1992), while the sequence extending between 1-2622 (Table 1) was not published.
A preliminary functional analysis of the promoter was performed by fusion of various deletions of the putative promoter sequence to the reporter gene uidA (GUS), and analysis of GUS expression (Jefferson et al.1987) in stable transgenic tomato plants. The results of the preliminary analysis were briefly reported (Carmi et al. 1994). Primer extension analysis indicated that the transcribed transcript of the TPRP-F1 gene includes 408 bp of untranslated leader sequence. A detailed analysis of the promoter region of the TPRP-F1 gene was performed including additional deletions and testing GUS expression in more organs and at additional developmental stages. The results of this analysis are summarized in Figure 1.
The main findings of this analysis are:
1. The sequence extending 2542 bp 5' to the translation initiation codon served as the promoter to which the rolB gene was fused in the chimeric gene, (see also Figs 2-8), Based on this analysis (see Fig. 1), a sequence extending only 1701bp 5' to the translation initiation codon is sufficient to drive ovary and developing embryo specific expression of the rolB gene (Table 2)
2. The results of this analysis also indicate that a promoter sequence extending 564 bp 5' to the translation initiation codon fused to a sequence extending from 915 to 2643 bp 5' to the translation initiation codon confers specificity to the ovary and young fruits and to developing embryos (Table 3). And this sequence too can be used to drive the expression of the rolB specifically in the fruit, leading to parthenocarpy withour the adverse effects of expression in other organs and additional developmental stages. Construction of the chimeric gene TPRP-F1::rolB
Construction of the plasmid pSB171
The recombinant plasmid pSB171 (Fig. 2) was obtained by subcloning the genomic
7kbp λG16 Hind III fragment into the Hind III site of pBluescript SK+ .
Construction of the plasmid pBSN28
The Nsi I internal fragment of plasmid pSB171 was isolated and subcloned into the
Pst I restriction site of pBluescript (Fig. 3)
Construction of the plasmid pBSN33
To ensure that the rolB gene will be translated from its native translation initiation codon, the translation initiation codon of the TPRP-F1 gene included in plasmid pBSN28 was abolished by in vitro mutagenesis: A PCR fragment containing the promoter with mutated initiation codon was prepared using the pBSN28 clone as a template, the sequence of the T7 promoter served as one primer (3' to 5') and the second primer was the following:
5' TGGTACCGGGCAATGAACAAAGTTCCA 3' . The bold letters designate the mutated sequence creating a new KpnI site at the 3' of the promoter sequence. The
PCR product was digested with KpnI and XbaI and ligated to pBluescript creating the plasmid pBSN33 (Fig. 4).
Construction of the plasmid pBSNrolB
pUC19-B26 (supplied by J. Schell) harbors the sequence between nucleotides 9814 and 11324 according to Slighton et al. (1986). The sequence of rolB included in this plasmid is given in Table 5. The EcoRI - HindIII fragment of this plasmid was isolated and cloned into the EcoRI/HindIII-restriction cleavage sites of the plasmid pBluescript to form pBSrolB (Fig. 5). The KpnI fragment of pBSrolB was isolated and subcloned into the KpnI site of pBSN33, and a clone with the rolB gene inserted in the correct orientation was identified by restriction enzyme analysis. This plasmid was named pBSNrolB (Fig. 6).
Construction of the binary plasmid pGB18rolB
The EcoRI-HindIII polylinker of plasmid pUC18 was isolated and subcloned into the EcoRI/HindIII-restriction cleavage sites of the plasmid pGA492 (An 1986) to form plasmid pGB18 (Fig. 7). The XbaI-BamHI fragment of pBSNrolB was isolated and subcloned into the XbaI/BamHI restriction cleavage sites of plasmid pGB18 to form plasmid pGB18rolB (Fig. 8).
Induction of Parthenocarpy in tomato via specific expression of the TPRP-F1: :rolB Gene in the ovary.
Plant transformation
The binary vector pGB18rolB was transformed into the indeterminate tomato breeding line MP-1 by co-cultivation of cotyledons derived from 10-day-old seedlings with Agrobacterium tumefaciens strain EAH105 (Hood et al. 1993). Essentially, the protocol described by McCormick (1991) was followed, except that the 'overnight' bacteria culture was resuspended in liquid MS (Murashige and Skoog 1962) medium before pouring over the cotyledons, and using 0.5 mg/L cefotaxime instead of carbenicillin. The cotyledons and the plantlets regenerated from them were propagated in the presence of 100 mg/L of kanamycin until rooted. Regenerated plants were planted in peat pellets (Jiffy-7, a/s Jiffy Products, Norway) for hardening (for 1-2 weeks) and then transferred to insect-proof greenhouse or net-house.
To select for transformed progenies, sterile seeds were germinated on selective medium (1/2 MS medium, 3% sucrose and 100 mg/L Kanamycin), where only transgenic seedlings develop a branched root system. Description of the transgenic plants
Three parthenocarpic primary (Ro) transgenic plants were regenerated from breeding line MP-1: MPB-4, MPB-12 and MPB-13, and MPB-19 . Transformation of the Ro plants was confirmed by PCR analysis, using primers for the selectable marker gene nptII, and by Southern analysis, using a KpnI fragment of the rolB gene as a probe.
The Southern analysis indicated that the MPB-4 Ro plant contains at least four inserts of the chimeric genes whereas both MPB-12 and MPB-13 contain two copies in tandem. The tandem pattern of insertion was confirmed by Southern analysis of R1 and R2 progenies of both MPB-12 and MPB-13.
Phenotypes of Ro Plants
Some phenotypic characteristics of the six Ro plants are summarized in Table 6.
1. Vegetative growth habit of Ro plants: Upon transplanting the Ro plants into pots, they developed a vegetative growth habit which did not differ significantly from that of the parental cultivar MP-1.
2. Reproductive development of the Ro plants: All the Ro plants developed multiple flowers. The flowers of plant MPB-4 manifested a typical 'parthenocarpic' mode of development namely, the ovaries started to enlarge very early relatively to the flower's development when the petals were still completely closed, and the petals did not wilt and remained attached to the base of the developing fruit until torn away by the growing fruit The MPB-4 pollen was not viable according to vital staining with fluorescein diacetate (Widholm 1972) and pollen germination test (Brewbaker & Kwack 1963).
The flowers of MPB-13-Ro plant manifested a moderate mode of parthenocarpic-like development (early enlargement of the ovary and postponed wilting of the petals), the pollen was viable. The flowers of MPB-12-Ro manifested even a milder parthenocarpic phenotype; their ovaries were slightly enlarged and wilting of the petals was postponed. Their pollen was viable. 3. Seedlessness and fruit characteristics of Ro plants: As summarized in Table 6 seedlessness was absolute in plant MPB-4, and in most of its fruits the columella was considerably larger than in the parental cultivar or in fruits of non- parthenocarpic transgenic plants. The locular cavities were full of jelly in most of the MPB-4 fruits, just in few the jelly fill was incomplete. Most of the fruits contained 4-5 locules whereas under the same growth conditions the MP-1 fruits usually consisted of 3-4 locules. Seeded fruits were not obtained from MPB-4 even following hand-pollination of very young flower buds with pollen of the parental line MP-1. This female sterility is apparently due to rapid enlargement of the ovary at very early stage of flower bud development leading to closure or detachment of the style tube which prevents fertilization. Thus, plant PMB-4 is considered an obligate parthenocarpic one. This indeterminate plant is maintained by propagation from cuttings.
Plants MPB-12 and MPB-13 behaved as facultative parthenocarpic ones. Their seedless fruits were full of jelly and contained 4 locules or more. In most MPB-13 fruits and in some of the MPB-12 fruits the columella was somewhat greater than in the parental line fruits. In some of the MPB-13 fruits the jelly was of greenish hue, especially in fruits developed at high temperatures. This phenomenon was also reported for auxin-induced fruits.(Abad & Monteiro 1989). The fasciation typical to auxin-induced parthenocarpic fruits was not observed in the seedless fruits of MPB-12 and MPB-13.
Among the seedless fruits of MPB-12-Ro developed in the summer, under extremely high temperatures, there was a tendency for a typical malformation; the fruits were oval rather than round, slightly flattened, and occasionally constricted in transverse section (a "peanut-like" appearance).
Phenotypes of R1 plants
1. Vegetative growth habit of R1 plants: Most of the Kanr seedlings of PMB-12-R1 and all of the Kanr MPB-13-R1 plants were characterized by broad cotyledons which rolled downwards. The severity of the phenomenon varied among the various plants. The root system of some of the MPB-13-R1 seedlings was more developed than that of the control plants. The developed plants did not manifest auxin related malformations (such as leaf epinasty, stem curling or adventitious root development.
2. Reproductive development of the R1 plants: Flowers of the various Kanr MPB- 12-R1 plants were of normal appearance and mode of development, except for the lack of wilting of the petals which remained attached to the base of the developing fruits, similar to the phenotype of the MPB-12-Ro flowers. Flowers of the Kanr MPB-13-R1 plants manifested varying degree of 'parthenocarpic' development (early enlargement of the ovary and postponed wilting of the petals), and the pollen was viable.
3. Seedlessness and fruit characteristics in R1 plants: As specified in Table 6, most of the fruits developed on the various Kanr MPB-12-R1 plants were seedless and full of jelly. Occasionally seeded fruits developed, most of them contained significantly less seeds than the parental cultivar, and the average weight and size of the seeds was significantly higher than that of the MP-1 fruits. The frequency of seeded fruits varied among the various R1 plants. The fruits were not puffy; some of the fruits were of slightly flattened oval shape. Transverse constriction was not observed, apparently because the fruits did not develop under exceptionally high temperatures. Most of the fruits of the various MPB-13-R1 plants were of regular shape, seedless, and full of jelly. Few seeded fruits developed, and most of them contained less seeds than MP-1.
Phenotypes of R2 plants
1. Vegetative growth habit of R2 plants : The phenomenon of broad cotyledons was also observed in the kanr R2 seedlings of MPB-12 and MPB-13. The developing plants were of normal growth habit.
2. Seedlessness and fruit characteristics in R2 plants: The fruits developed on R2 progenies of MPB-12 and MPB-13 transformants were tested for several fruit indexes. These plants grew under pollination-permissive environmental conditions. Data analysis was based on information collected from two or three plants of the R2 generation (Table 7), since no significant differences were found among the various R2 plants derived from a common R1 plant. The transgenic fruits did not differ significantly (P < 0.05) from the control fruits with regard to the average fruit weight number of locules per fruit or Brix value. However, even though pollination in the transgeneic plants was supported by vibrating the flowers, the fruits of all the transgenic plants had significantly (P < 0.001 ) fewer seeds than those of the control plants. The seed number varied from zero up to 30-50 per fruit among the various fruits developed on the same transgenic plant, reflecting the facultative nature of the parthenocarpy in these transgenic plants (Table 7 ).
In the parental line MP-1 there was a significant positive correlation between seed number and fruit weight (r2=0.386, P=0.023, n=14). However, the highly significant decrease of seed number in the transgenic plants was not accompanied by a decease of fruit weight. Regression of fruit weight vs. seed number was insignificant for MPB-12.5-R2 (n=23) r2=0.0279, P=0.446, for MPB-13.3-R2 (n=15), r2=0.0086, P=0.7419, and for MPB-13.4-R2 (n=7), r2=0.1115, P=0.4642. This fact indicates that the expression of the transgene completely compensates for the seed contribution to fruit weight.
All the seedless fruits were full of jelly. The color of jelly of the transgenic fruits was not always as red as that of the control; in many of the fruits it was more of yellow-orange color with traces of green hue.
It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described herein above. Rather the scope of the present invention is defined only by the claims which follow.

Claims

1. A method for the production of genetic parthenocarpy in plants comprising the steps of:
providing a cassette including;
a DNA sequence coding for modulation of auxin effects in plants; and
a promoter specific for the ovary between anthesis and early fruit development to control the DNA sequence;
introducing the cassette into a plant;
2. A method according to claim 1 and also comprising of the step of:
screening the plant for either facultative or obligatory parthenocarpic characteristics.
3. A method according to either claim 1 or claim 2 wherein the step of introducing comprises the steps of:
transformation of plant material;
and regeneration of transformed plants.
4. A method according to claim 3 wherein the plant material includes seed derived cotyledons.
5. A method according to claim 3 wherein the step of transformation includes the steps of:
providing a plasmid incorporating the cassette;
introducing the plasmid into A. tumerfaciens; and
incorporating the plasmid into the plant material by co-cultivation with the A. tumerfaciens including the plasmid.
6. A method according to claim 1 wherein the DNA sequence coding for modulation of auxin effects in plants includes the sequences of the rolB gene.
7. A method according to claim 1 wherein the promoter specific for the ovary between anthesis and early fruit production includes the sequences as specified in Table 1.
8. A method according to claim 1 wherein the promoter specific for the ovary between anthesis and early fruit production includes the sequences as specified in Table 2.
9. A method according to claim 1 wherein the promoter specific for the ovary between anthesis and early fruit production includes the sequences as specified in Table 3.
10. A method according to claim 1 wherein the promoter specific for the ovary between anthesis and early fruit production includes the sequences as specified in Table 4.
11. A method according to claim 2 wherein the step of screening includes:
growing the plant to produce fruit; and
examining the fruit for seed production under fertilization permissive conditions and/or under fertilization restrictive conditions.
12. A method according to claim 1 wherein the plant is a tomato plant
13. A tomato plant produced by the method of claim 1.
14. Fruit of the tomato plant of plant produced by the method of claim 1.
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US6268552B1 (en) 1997-05-06 2001-07-31 Kansas State University Research Foundation Transgenic seedless fruit comprising AGL or GH3 promoter operably linked to isopentenyl transferase or tryptophan monooxygenase coding DNA
EP0999738A4 (en) * 1997-05-06 2002-06-12 Univ Kansas State Transgenic seedless fruit and methods
EP1989306A1 (en) * 2006-02-13 2008-11-12 Adelaide Research & Innovation Pty Ltd. Plant egg cell transcriptional control sequences
EP1989306A4 (en) * 2006-02-13 2009-09-23 Adelaide Res & Innovation Pty Plant egg cell transcriptional control sequences
EP2405010A1 (en) * 2006-02-13 2012-01-11 Adelaide Research & Innovation Pty Ltd. Plant egg cell transcriptional control sequences
US8173864B2 (en) 2006-02-13 2012-05-08 Adelaide Research & Innovation Pty Ltd Plant egg cell transcriptional control sequences
US9139839B2 (en) 2006-02-13 2015-09-22 Adelaide Research & Innovation Pty Ltd Plant egg cell transcriptional control sequences

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EP1007704A2 (en) 2000-06-14
US6114602A (en) 2000-09-05
CN1213405A (en) 1999-04-07
TR199801573T2 (en) 1998-12-21
CA2246276A1 (en) 1997-08-21
IL117139A0 (en) 1996-06-18
AU1616297A (en) 1997-09-02
WO1997030165A3 (en) 1997-10-09

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