Genetic engineering of parthenocarpic fruit development in tomato

Transcription

1 Genetic engineering of parthenocarpic fruit development in tomato Tomato for the fresh market DefH9-iaaM gene is able to sustain parthenocarpic fruit development in two different tomato types (either cherry or round-shaped fruits). Fruit set percentage and fruit quality were not negatively affected in the primary transformants in conparison to selfed flowers of untrasformed control plants.

2 DefH9-iaaM gene is able to sustain parthenocarpic fruit development in two different tomato type (either cherry or round-shaped fruits). = selfed; EM= emasculated.

3 DefH9-iaaM is expressed in tomato flower buds

4 Percentage of fruit set CM #11 CM #12 CM #13 CM #14 CM Cont L.276 #1 L.276 #4 L.276 #6 L.276 #7 L.276 Cont genotypes Fruit set in emasculated flowers of GM DefH9-iaaM tomato plants Untransformed controls do not set fruits from emasculated flowers

5 Average fruit weight 140,0 120,0 100,0 Selfed Emasc. 80,0 g 60,0 40,0 20,0 0,0 CM #11 CM #12 CM #13 CM #14 CM Cont L.276 #1 L.276 #4 L.276 #6 L.276 #7 L.276 Cont genotypes Ficcadenti N., Sestili S., Pandolfini T., Cirillo C., Rotino G.L., and Spena, A. (1999) Genetic engineering of parthenocarpic fruit development in tomato. Mol. Breed. 5,

6 Greenhouse trial for evaluation of parthenocarpic transgenic hybrids (Spring Cultivation) Material 3 transgenic hybrids of round type tomato (one of them was F1 Giasone) 1 transgenic hybrid of cherry type tomato (experimental hybrid) Untransformed isogenic controls

7 Number of fruits/plant (early production) 25,0 cont rol transgenic 20,0 15,0 N 10,0 5,0 0,0 H F1 Giasone H.F H.F hybrids

8 N 40,0 35,0 30,0 25,0 20,0 15,0 10,0 5,0 0,0 Number of fruits/plant total prod control total prod transgenic H F1 Giasone H.F H.F genotypes

9 Average fruit weight (early prod) g H F1 Giasone H.F H.F hybrids control transgenic

10 Early production 3500 control transgenic g/plant H F1 Giasone H.F H.F Hybrids

11 Total Production control transgenic H F1 Giasone H.F H.F Hybrids

12 Giasone untransformed control Parthenocarpic DefH9-iaaM Giasone

13 Cherry type tomato Transgenic line = L CM iaam x L4 Control line = L CM x L4

14 Cherry tomato Number of fruits 60 transgenic 50 control 40 N I truss II truss I+II+III trusses total

15 Cherry tomato Average fruit weight 30 transgenic control N I truss II truss I+II+III trusses total

16 Cherry tomato Yield transgenic control g/plant I truss II truss I+II+III trusses total

17 Conclusions The DefH9-iaaM gene is able to sustain parthenocarpic fruit development in different types of tomato for the fresh market, Marketable fruits are produced under environmental conditions adverse for pollination and/or fertilization. No adverse effects on fruit quality Acciarri N., Ferrari V., Vitelli G., Ficcadenti, N., Pandolfini, T., Spena, A., and Rotino, G.L. (2000) Effetto della partenocarpia in ibridi di pomodoro geneticamente modificati. Informatore Agrario 4,

18 Genetic engineering of parthenocarpic fruit development in tomato Industrial tomato (The pickelhauben problem) In the cv UC82, a cultivar typical for processing tomatoes, the original gene DefH9-iaaM causes the production of parthenocarpic malformed fruits. The malformations are similar to those caused by an excess of exogenous auxin and/or an higher sensitivity of flowers to the hormonal treatment.

19 A genetic solution of the pickelhauben problem in tomato Control fruit GM malformed fruit Development of a genetic tool to reduce the expression and consequently the action of the DefH9-iaaM gene Solution: Downregulation of gene expression at the post-transcriptional level by replacing 53 b upstream the AUG initiation codon of the iaam gene with 87 b of the rola intron mutated at the splicing sites.

20 In vitro transcription translation analysis a) sequence of the mutated rola intron and scheme of the DefH9-RI-iaaM gene b) in vitro transcription-translation. Lane 1 and 2 iaam transcripts with different ULR. Lane 4 and 5, in vitro translation of iaam (lane 5) and RI-iaaM (lane 4). RI= reduced by intron

21 = selfed; EM= emasculated RT PCR analysis Estimated steady state level of mrna in tomato flower buds: DefH9-RI-iaaM: 1x10-8 of total mrna DefH9-iaaM: 1x10-7 of total mrna

22 Total IAA content in control and GM flower buds DefH9-iaaM: 100% (47/47) of the transgenic plants with malformed fruits DefH9-RI-iaaM: 10% (5/44) of the transgenic plants with malformed fruits IAA content in flower buds 30,00 25,00 20,00 nmol/g 15,00 10,00 5,00 0,00 ctr DefH9-RI-iaaM DefH9-iaaM genotypes

23 Average, maximun and minimum values in independent transgenic plants for DefH9-Ri-iaam (18 plants), DefH9- iaam (4 plants) and controls for percentage of fruit set from emasculated flowers and average fruit weight derived from emasculated and selfed flowers. Fruit set (%) Fruit weight (g) emasculated selfed aver min-max aver min-max RI-iaaM iaam control

24 Conclusions The modification of the 5 ULR of the DefH9-iaaM gene (i.e. DefH9-RI-iaaM gene) allows to produce high quality parthenocarpic fruits in industrial tomatoes (e.g. UC82). This method may solve the problem of a too high expressivity of a trait(s) when using transgenes affecting phytohormone synthesis. Pandolfini T., Rotino G.L., Camerini S., R. Defez, Spena A., Optimisation of transgene action at the post-transcriptional level: high quality parthenocarpic fruits in industrial tomatoes. BMC Biotechnology 2(1)