Two problems hampering efforts to produce salt-tolerant plants through constitutive expression of transgenes include: 1. Spatial control. Particular cell-types must respond specifically to salt stress to minimise the amount of Na⁺ delivered to the shoot; and, 2. Temporal control. Transgenes are typically expressed in plants at similar levels through time, irrespective of the stress encountered by the plant, which may exacerbate pleiotropic effects and means that, particularly in low-stress conditions, costly and/or detrimental metabolic processes may be active, thus reducing yield. To address these issues, Gateway® destination vector constructs were developed combining the GAL4 UAS (upstream activating sequence) with the ethanol-inducible gene expression system to drive inducible cell-specific expression of Na⁺ transporter transgenes (or to silence salt transporter transgenes inducibly and cell-specifically). Rice (Oryza sativa L. cv. Nipponbare) GAL4-GFP enhancer trap lines (Johnson et al., 2005: Plant J. 41, 779-789) that express GAL4 and GFP specifically in either the root epidermis or xylem parenchyma (and therefore ‘trap’ cell-type specific enhancer elements) were transformed with this GAL4 UAS – ethanol switch construct, thereby allowing both spatial and temporal control of transgenes. In preliminary experiments, the expression system successfully limited the expression of RFP to specific cell-types after induction with ethanol. Other genes expressed using this system include PpENA1, a Na⁺-extruding ATPase from the moss, Physcomitrella patens, and AtHKT1;1, a Na ⁺ transporter from Arabidopsis thaliana. The two enhancer trap rice lines were also transformed with the GAL4 UAS driving stable expression of AtHKT1;1 and PpENA1 specifically in root epidermal or xylem parenchyma cells. Expression of AtHKT1;1 in root epidermal cells reduced Na⁺ accumulation in the shoots, while expression in the root xylem parenchyma appeared to have little effect on shoot Na⁺ accumulation. Using cryo-scanning electron microscopy (SEM) X-ray microanalysis, the outer cells of the roots of the line expressing AtHKT1;1 in the epidermal cells were found to accumulate higher levels of Na⁺ than the parental enhancer trap line. Additionally, this line had decreased unidirectional ²²Na⁺ influx. Similar results were observed for plants expressing AtHKT1;1 driven by the CaMV 35S / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325289 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

[Truncated abstract] Lophopyrum elongatum, a wild relative of wheat, can be used as a source of novel genes for improving the salt tolerance of bread wheat. Na+ `exclusion? is a major physiological mechanism for salt tolerance in the wheat L. elongatum amphiploid, and a large proportion (~50%) of the improved Na+ `exclusion? is contributed by a gene(s) on chromosome 3E. This study integrated physiological analysis with comparative genomics to identify gene orthologues that may regulate Na+ transport, and designed and implemented molecular markers for developing wheat L. elongatum recombinant lines with reduced portions of L. elongatum chromatin retaining the Na+ `exclusion? trait. Physiological analysis of leaf Na+ accumulation in wheat L. elongatum substitution lines confirmed that the 3E chromosome contributes a major effect on reduced leaf Na+ accumulation in wheat when grown at 200 mM NaCl. Candidate genes from the model plant, Arabidopsis thaliana, controlling Na+ transport into and from cells (SOS1, HKT1) or compartmentalisation within vacuoles (NHX1, NHX5, AVP1, AVP2) were targeted for comparative analysis in wheat. Wheat gene orthologues were identified by BLAST searching to identify either FL-cDNAs or ESTs, which were subsequently used to amplify genomic DNA, and orthologues confirmed by similar intron-exon structure between Arabidopsis and rice. Intron-exon comparisons showed the majority of exons were conserved between Arabidopsis, rice and wheat, but also indicated exon shuffling events since divergence from a common ancestor. Gene orthologues were assigned to homoeologous chromosomes and non-syntenic regions between wheat and L. elongatum, with the SOS1 orthologue located on group 3 chromosomes in wheat and L. elongatum. ... The recombinant line 524-568 contains a small introgression on the distal end of the long arm of wheat chromosome 3A and represents the most desirable line presently available for further germplasm development. The main outcomes of this thesis have been an increased understanding of the physiology and evolution of orthologues for Na+ transport in wheat and L. elongatum, improved methodologies for designing alien-specific PCR markers, and the development of overlapping recombinant lines that provide a source of novel genes for pyramiding into wheat and improving its tolerance to salt stress.

Rice (Oryza sativa L.) is the principal crop of North Eastern Tanzania but production is threatened by salt affected soils, drought and the use of un-adapted cultivars, among other constraints. Little research and hardly any breeding have been done on the aspects of salt tolerance of the crop in sub Saharan Africa, leading to low yields and low production in rice irrigation schemes under arid and semi arid conditions. A project was therefore implemented in North Eastern Tanzania during 2007-2010 seasons to investigate the possible breeding contributions to enhance productivity and production of the crop in salt affected areas. The objective of this study was to: a) determine farmers’ perceptions on both salt problems and their effects on rice crop productivity as well as establishing farmers’ needs and preferences for rice varieties in the targeted irrigated environment; b) determine the extent of salt problem in both soil and irrigation water in the available rice irrigation schemes in the North-eastern Tanzania; c) identify the major physiological mechanisms associated with salt tolerance in farmer-preferred native varieties and landraces; and d) determine the mode of inheritance of salt tolerance in rice. Participatory rural appraisal was conducted in Mkomazi and Mombo villages in Tanga region with the aim of understanding characteristics of rice–based farm economy, farmers’ perception of agriculture constraints and variety preferences in salt affected areas of North-eastern Tanzania. This was followed by a preliminary study to understand soil characteristics in relation to salt problems and its extent in selected nine rice irrigation schemes. Studies was established under controlled conditions to assess the salt tolerance of some rice farmers preferred rice cultivar and evaluate the putative traits in the rice materials that contribute to the performance of a genotype under saline and saline-sodic condition. Thereafter, genetic mechanism governing various morpho-physiological parameters in selected Tanzania local farmers’ preferred varieties and salt tolerant donors under saline and sodic soil conditions of North Eastern Tanzania were determined. A participatory rural appraisal (PRA) established that rice was a major staple food and cash crop and rice farming was a major economic activity in the area. However, soil degradation through increased salt affected soils was identified as the major factor responsible for irrigated rice yield decline. Major varieties grown are salt sensitive, and salt tolerant varieties were not available. The study also revealed that most farmers’ preferred traits of rice cultivars were high yield potential, aroma, early maturing, medium plant stature, tolerance to salt and drought. Improvement of these characters in new salt tolerant varieties would increase food production in fields with low or zero productivity and the well-being of the poor farmers. A soil characterisation study indicated the magnitude of the problem, whereby, seven out of nine studied irrigation schemes were affected and sodic and saline-sodic conditions were the dominant types of soils. Poor irrigation canals and management of irrigation water were the driving factors that contributed to salts accumulation causing a decline in productivity. Experiments were established under controlled environments to evaluate the tolerance of 10 and 11 rice genotypes under saline and saline-sodic stresses, respectively. Significant variation between genotype and significant interactions between genotype and salt treatment (P<0.001) were observed for all characters studied. Genotype Pokkalli, IR 67076-2B-21-2 and IR 56 showed superior performance under saline, whereas CSR 27, Nerica 2 and IR 56 had superior performance under saline-sodic. The study therefore established that, all the local farmer preferred cultivars except IR 56 performed poorly under both salt stress environments. High seedling vigour, less leaf injury, less Na+ and high K+ accumulation in leaves, low Na+/K+ ratio of ion uptake, high spikelet fertility, increased grains per panicle and 1000 grain weight were considered as the desirable characteristics therefore can be used in developing lines for salt tolerance for production under saline and saline - sodic conditions. Gene action and combining ability studies for nine morpho-physiological traits were studied under normal, saline and sodic soil environments. The rice populations were generated through 7 x 7 full diallel crosses and advanced to F2. The parents comprised two donors for saline tolerance, one donor for sodic tolerance and four salt sensitive farmers preferred varieties. Both additive and non-additive gene effects were important in the inheritance of the characters studied in all soil environments. However, additive effects were more important for the number of tillers, shoot Na+, Na+/K+ ratio and plant height. Both additive and non-additive gene effects were important for spikelet fertility, days to 50% flowering, number of grains per panicle, 1000 grain weight, and grain yield; however the magnitude of additive gene effect was higher than non additive effects. Amongst the parental lines, the best general combiners for yield along with other traits were TXD 306 and IR 67076-2B-21-2 under normal non-saline/sodic condition; IR 56, Pokalli and TXD 306 under saline condition and CSR 27 and TXD 306 under sodic conditions. The overall results from this study indicated the possibility of improving both yield and salt tolerance from this set of germplasm; therefore contributing to increasing rice yields in the marginal salt affected environments. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

[Truncated abstract] In southern Australian farming systems the replacement of deep-rooted perennial native vegetation with shallow-rooted annual crops and pastures has resulted in rising groundwater tables and an increased incidence of dryland salinity. It has been suggested that to address this issue by restoring hydrological balance, large areas of agricultural land need to be vegetated with perennial plants. One of the most agriculturally productive ways to do this is to introduce perennial pastures, both into upslope groundwater