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Type: Thesis
Title: Cell type-specific manipulation of salt tolerance genes in wheat and barley.
Author: Krishnan, Mahima
Issue Date: 2013
School/Discipline: School of Agriculture, Food and Wine
Abstract: More than 67% of Australian cropping land is at risk of becoming saline and agriculture is increasingly utilising salt effected land (Rengasamy, 2002). Salinity has a significant impact on crop yield, and the identification and manipulation of genes that help to ameliorate yield penalties resulting from salinity can enhance agricultural production. Bread wheat, a hexaploid with AABBDD genome, has been long considered more salt tolerant than the tetraploid durum wheat with an AABB genome. The D genome, originally from Aegilops tauschii, contains a locus important for maintaining high K⁺/Na⁺, Kna1, on chromosome 4, which contains the HKT1;5 gene encoding a Na⁺ specific transporter, TaHKT1;5-D. The transcript of this gene was knocked down through RNAi. Plants containing the RNAi construct were found to accumulate higher levels of Na⁺ in the 4th leaf regardless of whether they were grown under control or mild salt stress conditions (75mM). This result supports previous findings that orthologues of HKT1;5 in other plants influence Na+ translocation from root to shoot (Ren et al., 2005; Davenport et al., 2007). The impact of TaHKT1;5 on salt tolerance was studied by subjecting transgenic plants to control or salt stress (75mM) conditions. Changes in phenotype were measured through non-destructive plant imaging (LemnaTec® Scanalyzer), but no phenotypic variation was observed as a result of the salt stress that was applied, suggesting the stress may have been too mild. In parallel with the knockdown approach, the HvHKT1;5 gene, an orthologue of the bread wheat Na⁺ transporter (TaHKT1;5-D), and a barley inorganic proton pyrophosphatase, HvHVP1, were overexpressed in barley through use of promoters thought to control cell type-specific expression. Promoters were identified through an MPSS database search for genes with low to moderate transcript levels and specificity for root-cortex or root-stele. The promoters controlling these genes were then isolated to drive HvHKT1;5 in root cortex and stele and HvHVP1 in root cortex. Four promoters were found to be promising: two stelar-specific and two cortex-specific and were placed upstream of HvHKT1;5 and HvHVP1. These constructs were then transformed into barley (cv. Golden Promise). Transgenic plants were grown in 100mM salt stress with two independent lines for each promoter:gene construct. Independent lines which included a stelar-specific promoter controlling HvHKT1;5 transcription showed reduced Na⁺ accumulation and increased K⁺ accumulation in 4th leaf xylem sap. Transgene mRNA was detected in both shoots and roots of the plant. In conclusion, while lowering levels of HKT1;5 transcript in wheat were not found to impact whole plant salinity tolerance, it did increase Na⁺ accumulation in the shoot. This was supported by the results in barley where overexpression of HvHKT1;5 resulted in lower Na⁺ levels and a concomitant increase in K⁺ levels in the shoot. Further study on whether this result impacts barley salt tolerance is currently underway.
Advisor: Jacobs, Andrew Keith
Tester, Mark Alfred
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2013
Keywords: salinity; cell type-specific expression; wheat; barley; HvHKT1;5; HvHVP1; TaHKT1;5-D
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