| Summary: | The 9-cis-epoxycarotenoid-dioxygenase (NCED) is a key regulatory enzyme in ABA biosynthesis in plants and its overexpression increases ABA levels that can increase water use efficiency (WUE). The use of the „super promoter‟ (sp) to drive an LeNCED1 transgene in tomato greatly improved WUE without affecting long-term plant growth but caused delayed seed germination and reduced rates of seedling establishment. The first aim of the present study was to generate useful, novel variation in NCED transgene expression by allowing an LeNCED1 transgene, driven by a histone H2A promoter (pH2A), to transpose to new positions in the genome. A stabilized activator element linked to a GUS marker gene (sAc) was used to allow transposition of an engineered dissociation element (Ds) containing the LeNCED1 transgene of format Ds1::pH2A::LeNCED1::Ds2 (T-DNA-Ds). In F1 (sAc × Ds) plants transposition was demonstrated by detection of empty donor site through PCR. The F2 generation was screened for stable integration of the Ds element and reduced gs. Three F3 families, namely 59F3, 102F3 and 116F3, were investigated: they showed multiple stable Ds transposition events, had low gs, and a range of growth rates. Genotype 102F5 had wild-type seed germination, higher bulk leaf ABA and xylem sap ABA and 60% higher gravimetric WUE (WUEp). However, 102F5 accumulated 32% less above ground dry weight than wild type under well-watered conditions 56 days after germination. The sAc and Ds approach allowed production of T-DNA-Ds and nptII kanamycin free 102F5 line which retained transposed Ds (Tr-Ds) elements. These progeny were cross pollinated with sAc to reactivate the transposition of the LeNCED1 transgene and should be an excellent material for generating further variation in ABA content. The second aim was to overcome the seed dormancy in high ABA, genotypes such as sp5 and sp12, by testing novel hydroxamic acid NCED inhibitors for their ability to stimulate germination and seedling establishment. Improvement in seed germination in sp12 was observed with two hydroxamic acid compounds, and an increase in seedling growth rate was also observed, although this was not statistically significant.
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