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Architecture engineering in the tomato


We have attempted to modify the tomato inflorescence in order to synchronize flower development. Constructs to modify expression of two key regulatory genes, FALSIFLORA (FALS) and SELF PRUNING (SP), have been made and transgenic plants with the desired phenotypes were obtained, albeit with accompanying developmental defects. Mapping of the An, wiry4 and S mutations has been achieved down to 1cM intervals, and the forthcoming tomato genome sequence should facilitate the identification of these genes in the near future.
We have investigated the role of photoreceptors such as phytochrome on the fruit-ripening process, with the objective of increasing tomato fruit quality. We have generated a series of phytochrome- and cryptochrome-deficient tomato mutants and have crossed them to make a range of different double, triple, and quadruple mutants. Many of these have very clear and dramatic effects on truss and fruit development. These have been the focus for assessing the role of individual photoreceptors in the fruit ripening process. We have grown these mutants in the greenhouse in Spain and have performed a series of analyses to look at general properties such as brix, viscosity, firmness, etc. In parallel these mutants have been grown under greenhouse conditions in Italy and RNA has been prepared from fruits harvested throughout the ripening process and used for cDNA-AFLP to identify differentially expressed genes. We have focused on the manipulation of key light signalling molecules. One objective was the isolation of constitutively photomorphogenic mutants in tomato. For this, we screened several thousand mutagenized seedlings in darkness. This approach was very successful and a range of new light signal transduction mutants with constitutive photomorphogenic phenotypes in darkness have been obtained. At least one of these is likely to be a null mutation in the tomato DET1 gene. A second objective has been the isolation of ATROVIOLACEA (ATV), DARK GREEN (DG), ANTHOCYANIN FRUIT (AF) and/or PUNCTATE (PN) genes from their respective mutants. We have performed basic physiological characterization of these mutants, which has been extended to photobiology experiments. These physiological characterizations have revealed that ATV and AF are the most interesting genes that warrant cloning. Most pogress has been made with ATV, which has been mapped to a small region on the long arm of chromosome 7. When combined, the atv and Af mutations result in fruits with extremely high levels of anthocyanins. Such a new kind of tomato fruit could be very interesting as a functional food, given the strong antioxidant activity of these compounds. A final objective involved the generation of tomato plants containing light hypersensitive versions of key light signalling intermediates such as DET1, COP1 and HY5. Most interestingly, DET1 has been suppressed specifically in fruits using fruit-specific promoters and RNA interference constructs, which resulted in the generation of normal healthy plants that produced fruits with significantly enhanced carotenoid and flavonoid contents.
We have attempted to eliminate side shoots by the judicial manipulation of LS gene expression in vegetative or floral meristems. Mutation of the LS gene causes inhibition of lateral shoot formation. However, ls mutants cannot be used commercially because the mutation also causes defects in flower development and so ls mutants have reduced fertility. Two general transgene-based approaches were pursued to overcome this: Complementation of the ls mutant using flower-specific LS constructs Inhibition of LS activity in vegetative meristems. The second approach has been the most promising. For this, we have successfully been able to inhibit LS activity using either RNAi constructs or a dominant negative version of LS with the 35S promoter. Both strategies result in tomato plants with normal flower development and fruit yields, but lacking lateral shoots. Furthermore, the phenotypes are inherited as dominant traits, implying that these transgenes-based methodologies could be employed in future breeding programmes.
We have utilized architecture genes to optimise leaf-inflorescence ratios. In particular, our work has focused on manipulation of the tomato KNOTTED-2 gene, a transcription factor. A large number of transgenic tomato plants with modulated expression of the TKN-2 gene have been generated and analysed. Most significantly it has been possible to generate plants with sessile leaves but normal flower inflorescences. As a further achievement we have now cloned the two-leaf gene from Lycopersicon pennellii.

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