Gossypium hirsutum, upland cotton, is one of the major crops grown in the United States and the world. Upland cotton is cultivated in areas that are ideal breeding grounds for the difficult to manage, southern root-knot nematode (RKN), Meloidogyne incognita. Host plant resistance is the most effective way to control RKN populations. However, resistance used in most breeding programs stems from a few related sources. Novel sources of resistance have been identified but have yet to be introduced into elite breeding lines or genetically studied. The objectives of this study are two-fold. The first is to develop elite germplasm by introgressing RKN resistance from primitive accessions into modern cotton genotypes via backcrossing. The second is to use simple sequence repeats (SSRs) to identify loci associated with RKN resistance in the primitive accessions. The genotypes used will be: 1) inoculated with M. incognita, 2) phenotypically analyzed by measuring the nematode reproduction as eggs per gram of fresh root and host response using a root gall index, 3) genetically evaluated by using SSR markers to detect polymorphisms between the RKN resistant TX accessions and DP90 (susceptible genotype), and 4) analyzed using linkage and mapping software. Elite germplasm that contains: 1) high yield potential and a high level of RKN- resistance or 2) high fiber quality and RKN-resistance was developed by performing two backcrosses based on phenotypic analyses. A third screen is currently underway to ensure the introgression of the RKN resistance genes. Agronomic tests will need to be done before the germplasm is released. Genetic analyses using SSR-based primer sets of the TX accessions did not yield expected results. Of the 508 primers sets tested, only 31 were polymorphic between the TX accessions and DP90. A bulked segregant analysis approach was used to test the 31 primer sets on the resistant and susceptible bulks of the F2 population but no polymorphisms were seen. However, analyses found that the TX accessions were more genetically similar to Mexico Wild Jack Jones than to Clevewilt 6-3-5. More work needs to be done to understand the mechanism of RKN resistance in the TX accessions.

The response of upland cotton (Gossypium hirsutum L.) to legume and small grain cover crop establishment, in-season nitrogen (N) rate, and fertilizer N placement was investigated in two experiments located in coastal plain Virginia and North Carolina. The first experiment examined 1) soil compaction and cotton yield response to strip-tillage compared to no-tillage with a precision planted tillage radish and 2) the influence of legume mix, rye, and legume mix/rye combination cover crops with four in-season nitrogen (N) rates applied to cotton on cover crop biomass, cover crop nutrient uptake, soil compaction, soil N cycling, petiole nitrate-N (NO3-N) during the first week of bloom, cotton lint yield, and fiber quality parameters over two years. Legume mix cover crops resulted in greater N uptake, soil NO3-N during the growing season, and lint yields compared to LMR, rye, and fallow treatments over both study years. Soil compaction and lint yields were not significantly different between strip-tilled and no-till with tillage radish treatments in either year. Relative lint yields after LM were maximized at 93% relative yield with 110 kg N ha-1 applied in-season while relative lint yields for cotton following LM with 0 kg N ha-1 applied reached 75%, measuring at least 9% higher than cotton following other cover crop treatments. The second experiment investigated the effect of five N rates (0, 45, 90, 135, and 180 kg N ha-1) and three placement methods (broadcast, surface banded, and injected) on lint yield, petiole nitrate-N (NO3-N), lint percent turnout, and fiber quality parameters. Nitrogen rate and placement had a significant effect on lint yield but only N rate affected petiole NO3-N concentration. It was estimated that injecting fertilizer N requires an N rate of 133 kg N ha-1 to achieve 95% relative yield while surface banded fertilizer N required a rate of 128 kg N ha-1 to produce 90% relative yield. A critical petiole NO3-N concentration threshold of 5,600 mg NO3-N kg-1 was calculated to reach 92% relative yield. Other agronomic management practices such as cover crop termination timing, cover crop species blends, and number of fertilizer N applications are of interest in order to develop better recommendations and promote conservation agricultural practices in coastal plain Virginia and North Carolina. / Master of Science / Upland cotton (Gossypium hirsutum L.) response to diverse species cover crop mixes, conservation tillage method, fertilizer N rate, and fertilizer N placement at side-dress was measured in two field studies conducted on the coastal plain soil in Virginia and North Carolina from 2016-2018. The objectives of the following research were to 1) examine the influence of two conservation tillage practices and four cover crop mixes on cover crop biomass production, soil compaction, cover crop nutrient uptake, soil N cycling, petiole nitrate (NO3-N) and cotton lint yield and 2) measure cotton performance in response to five N rate and three placement application methods. Legume mix (LM) cover crops contained more N in biomass, resulting in higher soil NO3-N during the growing season and higher lint yields at harvest compared to a legume mix and rye combination (LMR), rye, and fallow treatments. Soil compaction and lint yield were not significantly different between strip-tilled and no-till/tillage radish treatments in either year. Nitrogen rate and placement had a significant effect on lint yield but only N rate affected petiole NO3-N concentration. Injection of fertilizer N required an N rate of 133 kg N ha1 to achieve 95% relative yield while surface banded fertilizer N required a rate of 128 kg N ha-1 to produce 90% relative yield. A critical petiole NO3-N concentration threshold of 5,600 mg NO3-N kg-1 was also calculated to reach 92% relative yield. Future application of these results can include investigation of optimal N source for Virginia cotton production, best N placement method for cotton grown in high residue systems, and an economic analysis to determine optimum agronomic management for Virginia coastal plain cotton production.

Rop/Rac proteins are plant-specific monomeric guanosine triphosphate-binding proteins (G-proteins) with important functions in plant development. Until recently, only three cotton (Gossypium hirsutum) Rop/Rac G-protein genes were sequenced, representing subfamilies III and IV of the plant monomeric Gprotein family. In this project, members of subfamilies II and I were cloned, sequenced, and named GhRac2 and GhRac3, respectively. Using real-time reverse transcription PCR, expression of GhRac2 was highest during fiber elongation, decreasing significantly when cellulose biosynthesis began. Transcript abundance of GhRac3 doubled between fiber elongation and secondary wall synthesis, remaining constant until 20 days post-anthesis. Expression of GhRac2 and GhRac3 was compared between the unfertilized ovules of Gossypium hirsutum, Texas Marker 1 and two near-isogenic fiber-impaired mutants. Expression of GhRac2 and GhRac3 was significantly higher in wild type ovules than in Ligon lintless, a mutant impaired in fiber elongation, but was not different in Naked Seed, a mutant impaired in fiber initiation.

Upland cotton (Gossypium hirsutum ) is an important fiber crop grown throughout the southern United States. Plant-pathogenic nematodes are worm-like animals that feed on the roots of most agronomic crops, including cotton. The southern root-knot nematode (Meloidogyne incognita, RKN) and the reniform nematode (Rotylenchulus reniformis, RN) cause significant yield losses in cotton every year. Current sources of resistance are effective but limited, therefore historical screenings of cotton accessions were revisited in search for novel resistance sources. None were identified but many of the screened accessions possessed markers of known root-knot nematode and reniform nematode resistance. The emerging guava root-knot nematode (Meloidogyne enterolobii, GRKN) is a risk for upland cotton production, and identifying host plant resistance would greatly reduce the yield losses for growers. Assays were conducted on the currently available RN and RKN resistance sources inoculated with GRKN. No known nematode resistance gene suppressed GRKN infection, indicating that work must be done to protect crops from the eventual discovery of GRKN in Mississippi fields. Using the same resistance sources, tests were conducted to determine if the currently available resistances to RKN and RN offer any suppression of secondary infection of non-target nematode species for resistance. This is referred to as systemic acquired resistance, which is the induction of non-specific plant defense. Assays found that early inoculation with the nematode targeted by resistance did not effect infection by a secondary nematode species.

This research was focused on increasing the efficiency of upland cotton production in the northern cotton belt through the use of new fertilizer formulations, placement, and timings. The objectives of the experiments reported in this thesis were to: 1) evaluate the effects of side-dress potassium (K), sulfur (S), and boron (B) formulation and application timing on tissue nutrient levels during the bloom period; 2) evaluate lint yield response of cotton to different formulations of nitrogen (N), K, S and B applied at side-dress; and 3) compare 5x5 banding (5 cm beside and 5cm below the seed) and deep placement of complete N-P-K-S blends to current nutrient management strategies on early season plant growth, nodes above white flower, total nodes, petiole nutrient concentrations during bloom, and lint yield. Tissue S and B concentrations were increased more often than K concentrations when the nutrients were applied with side-dress N. When evaluating P and K placement, petiole P levels were found to be significantly higher in unfertilized plots when no side-dress N was applied. Phosphorus and K placement and/or rate had no effect on lint yield when N was applied at side-dress during the study. Environmental conditions potentially influenced the response to P and K placement as 5x5 placement produced yields significantly higher during 2013 growing season at location 1, while deep placement produced significantly higher yields in 2014 at location 3. As a result, Virginia nutrient management recommendations for cotton have been updated to incorporate management strategies to maximize lint yields. / Master of Science

Identifying the proper tillage system which provides the best agronomic benefits for cotton production in the coastal plain soils of Virginia was the basis for this research. Strip-tillage was evaluated from 2015-2016 on-farm to determine the effects of annual and biennial treatments on plant growth and lint yield, as well as measuring the impacts on soil compaction. Also, small plot tillage experiments were conducted from 2013-2016 assessing no-till, conventional tillage, minimum tillage, and strip-tillage as well as the subsequent effects of these systems on four cotton varieties. Biennial strip-tillage produced similar lint yields to annual strip-tillage at 3 of 4 locations, with only one location showing a significant difference in lint yield of 135 kg ha-1. Persistence of subsoil tillage within the row from the previous year was observed at some locations and plant heights were not different at all locations, although annual strip-tillage provided deeper potential rooting depths both early season and at harvest. In short term tillage systems, minimal penalties in plant growth and lint yield were observed in no-till verses the other systems, primarily associated with greater soil compaction, shorter plant heights, and lower yields. An overall 8% reduction in yield was found with no-till systems, with no significant differences in yield among tillage systems observed in any year. Varietal effects on plant growth and yield were observed annually, with FM 1944 GLB2 being the shortest plants, and DP 1321 B2RF having the tallest plants. No tillage by variety interaction was observed, supporting the idea that varieties respond similarly across tillage systems. / Master of Science

Upland cotton, Gossypium hirsutum L., is a natural source of fiber and a major row crop in the US with an estimated $7 billion raw product value in 2019. However, it is extremely sensitive to the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). With the evolution of herbicide-resistant weeds compounded by off-target spray damage on conventional cotton varieties outside the transgenic Enlist technology (Dow Agrosciences) of herbicide-tolerant cotton varieties (Dow Agrosciences), there is a need to identify and develop novel sources of herbicide tolerance gene for upland cotton genetic improvement. Cotton chromosome substitution (CS) lines carry introgressions from other cultivated and wild allotetraploid Gossypium species that could be sources of novel and exotic alleles for herbicide tolerance. A total of 50 CS lines of G. barbadense L. (CS-B), G. tomentosum Nuttal ex Seeman (CS-T), and G. mustelinum Meers ex Watt (CS-M), in the genetic background of G. hirsutum L. Texas Marker-1 (TM-1) were screened for resistance to a field-recommended rate (1.12 kg ae ha-1) of 2,4-D in the greenhouse. Seven CS lines, CS-T04-15, CS-B12, CS-B15sh, CS-T04, CS-B22sh, CS-T07, and CS-B04-15 with the lowest injury were evaluated for tolerance at four and seven weeks after seedling emergence under field conditions. Progeny tests conducted in the greenhouse validated 2,4-D tolerance of CS-B15sh, showing 41% lower injury than TM-1. Novel variants of CS-T04-15 and CS-T07 were identified with complete tolerance to the herbicide but are segregating. Uptake and translocation of 14C-labeled 2,4-D indicated that reduced translocation of 2,4-D may be the 2,4-D tolerance mechanism in CS-T04-15 and CS-T07, while gene(s) associated with metabolism and reduced auxin transport appeared associated with the 2,4-D tolerance in CS-B15sh. Transcriptome analysis revealed differential expression of genes in 2,4-D-treated CS-B15sh and TM-1 with several components of the 2,4-D/auxin response pathway, including ubiquitin E3 ligase, PB1|AUX/IAA, ARF transcription factors, and F box proteins of the SCFTIR1/AFB complex being up-regulated. Functional annotation of differentially expressed genes revealed down-regulation of auxin transport, suggesting a potential linkage with tolerance mechanism involving altered movement of 2,4-D in CS-B15sh. The selected highly tolerant cotton CS lines will need to be confirmed further using molecular assays.

Field studies were conducted during 2006 and 2007 to evaluate control of saw greenbriar and southern dewberry by various pasture herbicides and to assess forage tolerance of Tifton 85 bermudagrass to these herbicides. Herbicides evaluated in each study included triclopyr, picloram, 2,4-D, fluroxypyr, dicamba, aminopyralid, metsulfuron methyl and various combinations of the above. Visual ratings were taken on each herbicide efficacy experiment. Visual evaluations of phytotoxicity, measurements of dry matter yield, and forage quality were quantified for each of the bermudagrass tolerance trials. Saw greenbriar was best controlled at approximately one year after treatment by triclopyr at 10.9% ae v/v with diesel as the carrier (88-98%), although the lower rate of triclopyr + diesel at 0.87% ae v/v + 5% v/v and triclopyr alone at 0.87% ae v/v provided 49 to 86% control. Triclopyr + fluroxypyr at 0.25% ai v/v + 0.086% ai v/v gave best control of southern dewberry in both years when applied as an individual plant treatment (IPT) six weeks after shredding. In general, shredding 45 days prior to herbicide application gave an advantage to southern dewberry control versus not shredding. In 2006, triclopyr + fluroxypyr (IPT) was the only treatment to decrease Tifton 85 dry matter yield at the first harvest, with no effect observed at the second harvest. In 2007, both broadcast treatments containing triclopyr + fluroxypyr and the IPT treatment of triclopyr decreased dry matter yield at the first harvest, with triclopyr (IPT) being the only treatment to lower dry matter yield at the second harvest. Field studies were also conducted in 2006 and 2007 to assess sharppod morningglory control in Roundup Ready Flex® and LibertyLink® cotton systems. Herbicides evaluated included glyphosate, glufosinate, prometryn, fluometuron, and diuron. Visual ratings of percent weed control and sharppod morningglory plant counts were taken to assess control. Prometryn at 1.8 kg ai ha⁻¹ and fluometuron at 1.8 kg ai ha⁻¹ provided significant preemergence control (33-81%) of seedling sharppod morningglory. All rates of glyphosate (1.06 and 1.54 kg ai ha⁻¹) and glufosinate (0.45 and 0.6 kg ai ha⁻¹) controlled sharppod morningglory from 55 to 100% at both application timings. The addition of diuron at 1.12 kg ai ha⁻¹ to glyphosate and glufosinate at the late season application enhanced sharppod morningglory control by 3 to 16%. Additionally, in both years, no reduction in cotton yield was observed in the morningglory infested treatment when compared to the weed free treatment.

Doctor of Philosophy (PhD) / The Australian cotton industry has developed high yielding and high quality fibre production systems and attributes a significant contribution of this achievement to highly innovative breeding programs, specifically focused on the production of premium quality lint for the export market. Breeding programs have recently shifted attention to the development of new germplasm with superior stress tolerance to minimise yield losses attributed to adverse environmental conditions and inputs such as irrigation, fertilisers and pesticides. Various contributors to yield, such as physiology, biochemistry and gene expression have been implemented as screening tools for tolerance to high temperatures under growth cabinet and laboratory conditions but there has been little extension of these mechanisms to field based systems. This study evaluates tools for the identification of specific genotypic thermotolerance under field conditions using a multi-level ‘top down’ approach from crop to gene level. Field experiments were conducted in seasons 1 (2006) and 3 (2007) at Narrabri (Australia) and season 2 (2006) in Texas (The United States of America) and were supplemented by growth cabinet experiments to quantify cultivar differences in yield, physiology, biochemical function and gene expression under high temperatures. Whole plants were subjected to high temperatures in the field through the construction of Solarweave® tents and in the growth cabinet at a temperature of 42 oC. The effectiveness of these methods was then evaluated to establish a rapid and reliable screening tool for genotype specific thermotolerance that could potentially improve the efficiency of breeding programs and aid the development to high yielding cultivars for hot growing regions. Cotton cultivars Sicot 53 and Sicala 45 were evaluated for thermotolerance using crop level measurements (yield and fibre quality) and whole plant measurements (fruit retention) to determine the efficacy of these measurements as screening tools for thermotolerance under field conditions. Sicot 53 was selected as a relatively thermotolerant cultivar whereas Sicala 45 was selected as a cultivar with a lower relative thermotolerance and this assumption was made on the basis of yield in hot and cool environments under the CSIRO Australian cotton breeding program. Yield and fruit retention were lower under tents compared with ambient conditions in all 3 seasons. Yield and fruit retention were highly correlated in season 1 and were higher for Sicot 53 compared to Sicala 45 suggesting that fruit retention is a primary limitation to yield in a hot season. Thus yield and fruit retention are good indicators of thermotolerance in a hot season. Temperature treatment and cultivar differences were determined for fibre quality in seasons 1 and 3; however, quality exceeded the industry minimum thereby indicating that fibre quality is not a good determinant of thermotolerance. Physiological determinants of plant functionality such as photosynthesis, electron transport rate, stomatal conductance and transpiration rate were determined for cultivars Sicot 53 and Sicala 45 under the tents and an index of these parameters was also analysed to determine overall plant physiological capacity in the field. Physiological capacity was also determined under high temperatures in the growth cabinet using a light response curve at various levels of photosynthetically active radiation (PAR). Photosynthesis and electron transport rate decreased, whilst stomatal conductance and transpiration rate increased under the tents as well as under high temperatures in the growth cabinet. Photosynthesis and electron transport rate were higher for Sicot 53 but stomatal conductance and transpiration rate were higher for Sicala 45 under the tents. No cultivar differentiation was evident for plants grown under high temperatures in the growth cabinet. Temperature treatment and cultivar differences in physiological function were greater in a hot year (season 1), thereby indicating the importance of cultivar selection for thermotolerance in the presence of stress. Electron transport rate was correlated with yield in season 1, thus suggesting the suitability of this method for broad genotypic screening for thermotolerance under field conditions. Biochemical processes such as membrane integrity and enzyme viability were used to determine cultivar specific thermotolerance under high temperature stress in the laboratory, field and growth cabinet. Electrolyte leakage is an indicator of decreased membrane integrity and may be estimated by the relative electrical conductivity or relative cellular injury assays. The heat sensitivity of dehydrogenase activity, a proxy for cytochrome functionality and capacity for mitochondrial electron transport, may be quantified spectrophotometrically. Cellular membrane integrity and enzyme viability decreased sigmoidally with exposure to increasing temperatures in a water bath. Membrane integrity was higher for Sicot 53 compared with Sicala 45 under the tents and under high temperatures in the growth cabinet. No temperature treatment or cultivar differences were found for enzyme viability under the tents; however, enzyme viability for Sicala 45 was higher in the growth cabinet compared with Sicot 53. Relative electrical conductivity was strongly correlated with yield under ambient field conditions and under the tents, suggesting impairment of electron flow through photosynthetic and/or respiratory pathways, thus contributing to lower potential for ATP production and energy generation for yield contribution. Thus, the membrane integrity assay was considered to be a rapid and reliable tool for thermotolerance screening in cotton cultivars. Gene expression was examined for cultivars Sicot 53 and Sicala 45 grown under high (42 oC) temperatures in the growth cabinet. Rubisco activase expression was quantified using quantitative real-time polymerase chain reaction analysis and was decreased under high temperatures and was lower for Sicala 45 than Sicot 53. Maximum cultivar differentiation was found after 1.0 h exposure to high temperatures and hence, leaf tissue sampled from this time point was further analysed for global gene profiling using cDNA microarrays. Genes involved in metabolism, heat shock protein generation, electron flow and ATP generation were down-regulated under high temperatures in the growth cabinet and a greater number of genes were differentially expressed for Sicala 45, thereby indicating a higher level of heat stress and a greater requirement for mobilisation of protective and compensatory mechanisms compared with Sicot 53. Cultivar specific thermotolerance determination using gene profiling may be a useful tool for understanding the underlying basis of physiological and biochemical responses to high temperature stress in the growth cabinet. There is future opportunity for profiling genes associated with heat stress and heat tolerance for identification of key genes associated with superior cultivar performance under high temperature stress and characterisation of these genes under field conditions. This research has identified cultivar differences in yield under field conditions and has identified multiple physiological and biochemical pathways that may contribute to these differences. Future characterisation of genes associated with heat stress and heat tolerance under growth cabinet conditions may be extended to field conditions, thus providing the underlying basis of the response of cotton to high temperature stress. Electron transport rate and relative electrical conductivity were found to be rapid and reliable determinants of cultivar specific thermotolerance and hence may be extended to broad-spectrum screening of a range of cotton cultivars and species and under a range of abiotic stress. This will enable the identification of superior cotton cultivars for incorporation into local breeding programs for Australian and American cotton production systems.

Doctor of Philosophy (PhD) / The Australian cotton industry has developed high yielding and high quality fibre production systems and attributes a significant contribution of this achievement to highly innovative breeding programs, specifically focused on the production of premium quality lint for the export market. Breeding programs have recently shifted attention to the development of new germplasm with superior stress tolerance to minimise yield losses attributed to adverse environmental conditions and inputs such as irrigation, fertilisers and pesticides. Various contributors to yield, such as physiology, biochemistry and gene expression have been implemented as screening tools for tolerance to high temperatures under growth cabinet and laboratory conditions but there has been little extension of these mechanisms to field based systems. This study evaluates tools for the identification of specific genotypic thermotolerance under field conditions using a multi-level ‘top down’ approach from crop to gene level. Field experiments were conducted in seasons 1 (2006) and 3 (2007) at Narrabri (Australia) and season 2 (2006) in Texas (The United States of America) and were supplemented by growth cabinet experiments to quantify cultivar differences in yield, physiology, biochemical function and gene expression under high temperatures. Whole plants were subjected to high temperatures in the field through the construction of Solarweave® tents and in the growth cabinet at a temperature of 42 oC. The effectiveness of these methods was then evaluated to establish a rapid and reliable screening tool for genotype specific thermotolerance that could potentially improve the efficiency of breeding programs and aid the development to high yielding cultivars for hot growing regions. Cotton cultivars Sicot 53 and Sicala 45 were evaluated for thermotolerance using crop level measurements (yield and fibre quality) and whole plant measurements (fruit retention) to determine the efficacy of these measurements as screening tools for thermotolerance under field conditions. Sicot 53 was selected as a relatively thermotolerant cultivar whereas Sicala 45 was selected as a cultivar with a lower relative thermotolerance and this assumption was made on the basis of yield in hot and cool environments under the CSIRO Australian cotton breeding program. Yield and fruit retention were lower under tents compared with ambient conditions in all 3 seasons. Yield and fruit retention were highly correlated in season 1 and were higher for Sicot 53 compared to Sicala 45 suggesting that fruit retention is a primary limitation to yield in a hot season. Thus yield and fruit retention are good indicators of thermotolerance in a hot season. Temperature treatment and cultivar differences were determined for fibre quality in seasons 1 and 3; however, quality exceeded the industry minimum thereby indicating that fibre quality is not a good determinant of thermotolerance. Physiological determinants of plant functionality such as photosynthesis, electron transport rate, stomatal conductance and transpiration rate were determined for cultivars Sicot 53 and Sicala 45 under the tents and an index of these parameters was also analysed to determine overall plant physiological capacity in the field. Physiological capacity was also determined under high temperatures in the growth cabinet using a light response curve at various levels of photosynthetically active radiation (PAR). Photosynthesis and electron transport rate decreased, whilst stomatal conductance and transpiration rate increased under the tents as well as under high temperatures in the growth cabinet. Photosynthesis and electron transport rate were higher for Sicot 53 but stomatal conductance and transpiration rate were higher for Sicala 45 under the tents. No cultivar differentiation was evident for plants grown under high temperatures in the growth cabinet. Temperature treatment and cultivar differences in physiological function were greater in a hot year (season 1), thereby indicating the importance of cultivar selection for thermotolerance in the presence of stress. Electron transport rate was correlated with yield in season 1, thus suggesting the suitability of this method for broad genotypic screening for thermotolerance under field conditions. Biochemical processes such as membrane integrity and enzyme viability were used to determine cultivar specific thermotolerance under high temperature stress in the laboratory, field and growth cabinet. Electrolyte leakage is an indicator of decreased membrane integrity and may be estimated by the relative electrical conductivity or relative cellular injury assays. The heat sensitivity of dehydrogenase activity, a proxy for cytochrome functionality and capacity for mitochondrial electron transport, may be quantified spectrophotometrically. Cellular membrane integrity and enzyme viability decreased sigmoidally with exposure to increasing temperatures in a water bath. Membrane integrity was higher for Sicot 53 compared with Sicala 45 under the tents and under high temperatures in the growth cabinet. No temperature treatment or cultivar differences were found for enzyme viability under the tents; however, enzyme viability for Sicala 45 was higher in the growth cabinet compared with Sicot 53. Relative electrical conductivity was strongly correlated with yield under ambient field conditions and under the tents, suggesting impairment of electron flow through photosynthetic and/or respiratory pathways, thus contributing to lower potential for ATP production and energy generation for yield contribution. Thus, the membrane integrity assay was considered to be a rapid and reliable tool for thermotolerance screening in cotton cultivars. Gene expression was examined for cultivars Sicot 53 and Sicala 45 grown under high (42 oC) temperatures in the growth cabinet. Rubisco activase expression was quantified using quantitative real-time polymerase chain reaction analysis and was decreased under high temperatures and was lower for Sicala 45 than Sicot 53. Maximum cultivar differentiation was found after 1.0 h exposure to high temperatures and hence, leaf tissue sampled from this time point was further analysed for global gene profiling using cDNA microarrays. Genes involved in metabolism, heat shock protein generation, electron flow and ATP generation were down-regulated under high temperatures in the growth cabinet and a greater number of genes were differentially expressed for Sicala 45, thereby indicating a higher level of heat stress and a greater requirement for mobilisation of protective and compensatory mechanisms compared with Sicot 53. Cultivar specific thermotolerance determination using gene profiling may be a useful tool for understanding the underlying basis of physiological and biochemical responses to high temperature stress in the growth cabinet. There is future opportunity for profiling genes associated with heat stress and heat tolerance for identification of key genes associated with superior cultivar performance under high temperature stress and characterisation of these genes under field conditions. This research has identified cultivar differences in yield under field conditions and has identified multiple physiological and biochemical pathways that may contribute to these differences. Future characterisation of genes associated with heat stress and heat tolerance under growth cabinet conditions may be extended to field conditions, thus providing the underlying basis of the response of cotton to high temperature stress. Electron transport rate and relative electrical conductivity were found to be rapid and reliable determinants of cultivar specific thermotolerance and hence may be extended to broad-spectrum screening of a range of cotton cultivars and species and under a range of abiotic stress. This will enable the identification of superior cotton cultivars for incorporation into local breeding programs for Australian and American cotton production systems.