Parthenocarpy. Production of fruit in absence of fertilization. Fruits are SEEDLESS. Seedlessness is advantageous for: consumers growers

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1 Parthenocarpy Production of fruit in absence of fertilization Fruits are SEEDLESS Seedlessness is advantageous for: consumers growers

2 Tools for seedless fruit production Genetics: mutants (cucumber, tomato, eggplant, grape, etc) alteration of ploidy (watermelon, banana) Problems: phenotypic side-effects polygenic genetic base Agronomical: spraying flowers with plant growth regulators tomato, eggplant, zucchini (out-of-season production) grape (fruit set and fruit growth) Drawbacks: costly, labour intensive, weather dependent

3 Genetic engineering parthenocarpy Endogenous increment of auxin in the ovary (ovule and/or carpel) EcoRI KpnI SphI HindIII DefH9 regulatory sequence iaam coding region Nos DefH9-iaaM

4 The DefH9 regulatory sequences drive expression in the placenta-ovules

5 Genetic engineering of parthenocarpy in eggplant Untransformed control GM DefH9-iaaM eggplant

6 Parthenocarpic fruits Effect of the DefH9-iaaM gene in a genetic background with a parthenocarpic tendency: Control = Tal1/1 untransformed Tal1/1 iaam=gm with the DefH9-iaaM gene DefH9-iaaM eggplant fruits from either emasculated or from self pollinated flowers

7 Conclusion DefH9-iaaM gene confers parthenocarpic fruit development (facultative parthenocarpy) Transgenic marketable parthenocarpic fruits are obtained under enviromental condition prohibitive for untransformed control plants (proof of concept) Rotino G.L., Perri E., Zottini M., Sommer H., and Spena A. (1997) Genetic engineering of parthenocarpic plants. Nature Biotech. 15,

8 Greenhouse winter production of transgenic parthenocarpic hybrids Trial carried out at Vittoria (RG), Sicily Materials Transgenic hybrids: : P1, P3 and P4 Untrasformed hybrids: : C1, C2 and cv Talina F1 C1 ctr P1 C2 ctr P3 and P4 Plastic (PE)-wood greenhouse

9 Methods Randomized block design 6 replicas (3 sprayed and 3 not sprayed) Plot 5 mq; 2 plants/mq P.g.r. Sedlene Melanzana (0.5 GA % β-noa) sprayed once a week on open flowers Normal agronomic practice Data collected: Yield and N N of fruit (on 5 plants/plot) Air and soil temperature; light intensity

10 35 Min. Max. Aver. 30 Temperature during the winter trial C nov 28-nov 12-dic 26-dic 9-gen 23-gen 6-feb 20-feb 6-mar 20-mar 3-apr 17-apr 1-mag 15-mag 29-mag GM fruit growth is enhanced L= untransformed not sprayed c=untransformed sprayed r= GM not sprayed l c r

11 Winter production sprayed not sprayed g/plant P1 C1 P3 P4 C2 Tal F1 Genotypes C1=untransformed control of P1 C2= untransformed control of P3 and P4 P1, P3, P4: DefH9-iaaM eggplants TalF1= genetic background with parthenocarpic tendency

12 Total Production (February-may) sprayed not sprayed 5000 g/plant P1 C1 P3 P4 C2 Tal F1 Genotypes

13 Number of fruits/plant Winter production sprayed not sprayed N P1 C1 P3 P4 C2 Tal F1 Genotypes

14 Number of fruits/plant Total production sprayed not sprayed N P1 C1 P3 P4 C2 Tal F1 Genotypes

15 HARVEST GM not sprayed Control not sprayed GM sprayed Control sprayed

16 Conclusion Transgenic hybrids give a significantly higher production than controls sprayed with phytohormones No need of phytohormones spray Reduction of 9% of the estimated total cultivation costs GM seedless eggplant fruit Donzella. G., Spena A. and Rotino, G.L. (2000) Transgenic parthenocarpic eggplants: superior germplasm for increased winter production. Mol. Breed. 6,

17 Greenhouse early spring and open field production of transgenic parthenocarpic eggplant hybrids

18 Greenhouse locations: Monsampolo del Tronto (AP) Pontecagnano (SA) Open field location : Monsampolo del Tronto (AP) Materials Greenhouse: Transgenic parthenocarpic hybrids (P1; P3; P5) were compared with corresponding controls (C1; C2) and cultivar Talina (SG-Sandoz) Open field: Transgenic hybrids P1 (long shape) and P10 (oval shape) were compared with corresponding controls C1 and C10

19 Greenhouse : early production per plant, average of two locations g P1 C1 P2 P5 C2 Talina Hybrids

20 Greenhouse: total productionper plant a 2500 a a a g b b 0 P1 C1 P2 P5 C2 Talina Hybrids

21 Greenhouse n fruits/plant early production P1 C1 P2 P5 C2 Talina Hybrids

22 Greenhouse n fruits/plant total production P1 C1 P2 P5 C2 Talina Hybrids

23 Conclusions Greenhouse cultivation for early spring production DefH9-iaaM gene causes parthenocarpic fruit development Marketable parthenocarpic fruits are obtained under cultivation conditions prohibitive for control plants GM Hybrids have given a significantly higher production than controls Fruits are seedless Acciarri N, Restaino F., Vitelli G., Perrone D., Zottini M., Pandolfini T., Spena A., Rotino G.L. (2002) Genetically modified parthenocarpic eggplants: improved fruit productivity under both greenhouse and open field cultivation. BMC Biotechnology 2(4).

24 Open field: early production g /p lan t P1 C1 P10 C10 Hybrids

25 Open field: total production g/plant P1 C1 P10 C10 Hybrids

26 Open field: early production 3,0 2,5 2,0 n fruits/plant 1,5 1,0 0,5 0,0 P1 C1 P10 C10 Hybrids

27 Open field: total production 10,0 9,0 8,0 7,0 n fruits/plant 6,0 5,0 4,0 3,0 2,0 1,0 0,0 P1 C1 P10 C10 Hybrids

28 Conclusions Open field trial Higher early production Seedless fruits Better planning of the harvests High fruit quality for both fresh market and industry Acciarri N, Restaino F., Vitelli G., Perrone D., Zottini M., Pandolfini T., Spena A., Rotino G.L. (2002) Genetically modified parthenocarpic eggplants: improved fruit productivity under both greenhouse and open field cultivation. BMC Biotechnology 2(4).

29 Increment of Eq. diameter emasc././selfed 120 % of selfed fruit Tal 1-1 Ctr Em Tal F1 Ctr Em Tal 1-1 #1-1 Em Days after fruit set Growth of GM pollinated and emasculated fruits is identical Growth of Talina (TalF1) emasculated fruits is curtailed in comparison to pollinated fruits

30 Increment of Pol. diameter emasc././selfed % of selfed fruit Tal 1-1 Ctr Tal F1 Ctr Tal 1-1 # Days after fruit set Growth of GM pollinated and emasculated fruits is identical Growth of Talina (TalF1) emasculated fruits is curtailed in comparison to pollinated fruits

31 DefH9-iaaM gene is expressed during both flower and fruit development This finding is consistent with the faster growth of parthenocarpic fruits

32 How do we obtain F1 transgenic parthenocarpic seeds? All the trials were performed with the DefH9-iaaM gene at the hemizygous state DefH9-iaaM was expressed and gave satisfactory results when inserted (by cross pollination) in different genetic background Seeds have been obtained from aged DefH9-iaaM transgenic plant The delayed female fertility allows to produce homozygous lines by selfing Homozygous lines for DefH9-iaaM are employed as male parents for mass multiplication

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