Novel promoters that induce specific transgene expression during the green to ripening stages of tomato fruit development.

UNLABELLED: Fruit-specific promoters have been used as genetic engineering tools for studies on molecular mechanism of fruit development and advance in fruit quality and additional value by increasing functional component. Especially fruit-ripening specific promoters have been well utilized and studied in tomato; however, few studies have reported the development of promoters that act at fruit developing stages such as immature green and mature green periods. In this study, we report novel promoters for gene expression during the green to ripening stages of tomato fruit development. Genes specifically expressed at tomato fruit were selected using microarray data. Subsequent to confirmation of the expression of the selected 12 genes, upstream DNA fragments of the genes LA22CD07, Les.3122.2.A1_a_at and LesAffx.6852.1.S1_at which specifically expressed at fruit were isolated from tomato genomic DNA as promoter regions. Isolated promoter regions were fused with the GUS gene and the resultant constructs were introduced into tomato by agrobacterium-mediated transformation for evaluation of promoter activity in tomato fruit. The two promoters of LA22CD07, and LesAffx.6852.1.S1_at showed strong activity in the fruit, weak activity in the flower and undetectable activity in other tissues. Unlike well-known fruit-ripening specific promoters, such as the E8 promoter, these promoters exhibited strong activity in green fruit in addition to red-ripening fruit, indicating that the promoters are suitable for transgene expression during green to ripening stages of tomato fruit development. KEY MESSAGE: Novel fruit-specific promoters have been identified and are suitable for transgene expression during green to ripening stages of tomato fruit development.

Molecular breeding of tomato lines for mass production of miraculin in a plant factory.

A transgenic tomato line (56B, "Moneymaker") that expresses the miraculin gene driven by the CaMV 35S promoter was crossed with a dwarf tomato ("Micro-Tom") for the molecular breeding of cultivars that are suitable for miraculin production in a closed cultivation system. Plant size, miraculin accumulation, and self-pruning growth were used as selection indicators for F2 plants. Two lines were chosen for further analysis, bred to the F6 or F7 generation and cultivated in a closed cultivation system. In 56B and the two crossed lines, the concentrations of miraculin in the pericarp were 140, 367, and 343 microg/g FW, respectively. We also estimated that 26.2, 73.6, and 45.9 kg FW/m2 of tomatoes and 2.2, 16.6, and 9.8 mg/m2 of miraculin in the pericarp, respectively, could be harvested per year. These two crossed lines will be useful for the mass production of miraculin, especially in a closed cultivation system.