Tomato is one of the most important horticultultural crops and represents an important source of vitamins, minerals, and antioxidants. Intragenic methods for tomato were first applied to redesign Calgene's "FlavrSavr". The transgenic crop contained an extended Agrobacterium T-DNA region with an extra copy of the polygalacturonidase (Pg) gene inserted in the antisense orientation between the 35S promoter of cauliflower mosaic virus and the terminator of the Agrobacterium tml gene. It also carried a bacterial expression cassette for the neomycin phosphotransferase (nptII) gene. Despite its extended shelf life, opposition from nongovernmental organizations resulted in an eventual withdrawal of FlavrSavr tomatoes from the market.
The intragenic version of the extended-shelf life concept was developed by first creating a silencing construct comprising Pg gene fragments inserted as an inverted repeat between convergently oriented promoters. The marker-free transfer DNA used to introduce this construct into tomato contained two copies of the 25-bp border-like Le02 element (Rommens et al. 2005), positioned as a direct repeat. This element is more effective as right border than either Le01 or Le03 in supporting tobacco transformation when linked to a 164-bp fragment from tomato AY850394 (nucleotides 42723-42886). The complementing left border region was created by fusing the second copy of Le02 to a 189-bp AT-rich region similar to AP009548 (reverse sequence of 10345-10532). A binary vector carrying both the left and right tomato-derived border regions, designated pSIM894, was used for marker-free transformation to incorporate the new quality trait.
Future applications may support efforts to unleash the full quality potential of tomato. For instance, tomato plants evolved to produce high levels of antioxidant flavonols in anthers and pollen only. Replacement of the promoter of the chalcone isomerase (Chi) gene by a fruit-specific promoter extended flavonol production to the edible parts of tomato (Muir et al. 2001). There is a variety of promoters that can be used to direct gene expression to fruit tissues. These promoters include the ethylene-responsive fruit-ripening E8 gene (Deikman et al. 1992) and the fruit-specific 2A11 gene (Van Haaren and Houck 1993). The most frequently used promoter for near-constitutive expression was isolated from the tomato ubiquitin-3 (tUbi3) gene (Hoffman et al. 1991).
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