A fifth of the calories consumed by humans come from wheat. In 2023, 789 million tonnes of wheat were produced worldwide. With an increasing human population, demand for wheat is expected to increase. With rising production costs and increased environmental instability, novel solutions are required to enhance productivity. Beneficial fungal endophytes belonging to the Epichloë genus may provide a solution. These endophytes are used as protection against insect herbivory in commercial pasture grasses throughout the world via animal safe secondary metabolites. Other benefits from Epichloë include resistance to fungal pathogens and increased resistance to abiotic stress such as drought. Asexual Epichloë, which are used in commercial forage grasses, grow intercellularly and are vertically transmitted (maternally inherited). Modern wheat could benefit from this technology by reducing the synthetic chemistry requirements for control of biotic stress. However, to date there are no Epichloë found naturally in symbiosis with modern wheat cultivars and establishing symbioses has been a challenge. Early work successfully involved Chinese spring-based wheat lines that contained alien chromosomes from wild grasses known to host Epichloë endophytes. One of the more successful associations was with TACBOW0011, which has a Leymus racemosus H chromosome substitution. However, there were several negative phenotypes associated with endophyte colonisation, which included stunting, delayed flowering, shrivelled seed, low germination and low endophyte transmission. Through traditional breeding methods, improvements in plant phenotypes compatible with Epichloë endophytes will be described. Progress includes improved plant height, reduced flowering delay, increased seed size and improved germination and endophyte transmission.

Anthesis prediction is crucial for breeding wheat. While current tools provide estimates of average anthesis at the field scale, they fail to address the needs of breeders who require accurate predictions for individual plants. Hybrid breeders have to finalize their plans for pollination at least 10 days before such flowering is due and biotechnology field trials in the United States and Australia must report to regulators 7-14 days before the first plant flowers. Currently, predicting anthesis of individual wheat plants is a labour-intensive, inefficient, and costly process. Individual wheat of the same cultivar within the same field may exhibit substantial variations in anthesis timing, due to significant variations in their immediate surroundings. In this study, we developed an efficient and cost-effective machine vision approach to predict anthesis of individual wheat plants. By integrating RGB imagery with in-situ meteorological data, our multimodal framework simplifies the anthesis prediction problem into binary or three-class classification tasks, aligning with breeders' requirements in individual wheat flowering prediction on the crucial days before anthesis. Furthermore, we incorporated a few-shot learning method to improve the model's adaptability across different growth environments and to address the challenge of limited training data. The model achieved an F1 score above 0.8 in all planting settings.

Field trials play an essential role in developing genetically modified and genome-edited biotechnology plants, as they assess plant growth, yield, and potential unintended effects. Australian biotechnology field trials are regulated by federal protocols that mandate accurate forecasting of flowering times. Currently, this relies on labour-intensive and subjective visual field inspections of individual wheat plants at defined growth stages (Zadoks growth stages Z37, Z39, and Z41). To enable automatic forecasting, hyperspectral and red-green-blue (RGB) images were captured in the greenhouse, and hyperspectral reflectance data were acquired in a semi-natural environment. Support Vector Machine classification achieved F1 scores (0.832) for pre-anthesis growth stage classification through the combined use and systematic comparison of three spectral transformations, including Standard Normal Variate, Hyper-hue, or Principal Component Analysis, which together outperformed reliance on any single transformation. After feature selection, F1 scores (0.752) could be achieved with only five wavelengths. Furthermore, the SNV transformation demonstrated robust performance under limited training conditions, maintaining high classification accuracy and strong generalizability across varying data sizes. These findings highlight the effectiveness of transformation-enriched data and optimized feature selection for accurate growth stage classification, providing a low-cost approach to reduce manual inspection burdens and strengthen biosafety during biotechnology field trial practices.

Salt contamination of soils and irrigation water is a significant environmental concern for crop production. Leaf sodium (Na+) exclusion is commonly proposed to be a key subtrait of salt tolerance for many crop plants. High-Affinity Potassium (K+) Transporter 1 (HKT1) proteins have previously been identified as major controllers of leaf Na+ exclusion across diverse species. However, leaf Na+ exclusion does not always correlate with salt tolerance. We discuss literature which shows leaf Na+ accumulation can, in some circumstances, be tolerated without a detrimental effect on yield when HKT1 still functions to exclude Na+ from reproductive tissues. We conclude that, by having an ultimate role in the protection of reproductive performance, HKT1s' role in adaptation to salinity warrants redefinition.

KEY MESSAGE:The dwarfing allele Rht14 of durum wheat associates with greater stigma length, an important trait for hybrid breeding, whilst major dwarfing alleles Rht-B1b and Rht-D1b showed little to no effect. Although much understudied in wheat, the stigma is a crucial component for attaining grain set, the fundamental basis for yield, particularly in hybrid production systems where successful grain set relies on wind-driven pollen dispersal by the male parent and effective pollen capture by the female parent. Females with long stigma that exsert early are thought to be advantageous. Using glasshouse-grown lines, we examined variation in Total Stigma Length (TSL) across diverse panels comprising 27 durum and 116 bread wheat genotypes. Contrasting genotypes were selected for population development and genetic analysis. Quantitative trait loci (QTL) analysis was performed on a durum F2 population and a bread wheat recombinant inbred line (RIL) population. Contrasting with studies of anther length, we found no large effect on TSL of the GA-insensitive semi-dwarfing genes Rht-B1 and Rht-D1 in either durum or bread wheat. However, in durum cultivar Italo, we identified a region on chromosome 6A which is robustly associated with larger TSL and contains the Rht14 allele for reduced plant height, a trait that is favourable for female line development in hybrid systems. This dual effect locus explained 25.2 and 19.2% of TSL phenotypic variation in experiments across two growing seasons, with preliminary results suggesting this locus may increase TSL when transferred to bread wheat. In a bread wheat, RIL population minor QTL on 1A and 2A was indicated, but the strongest association was with Ppd-B1. Methods developed here, and the identification of a TSL-enhancing locus provides advances and further opportunities in the study of wheat stigma.

To maintain sustainable broadacre farming in the future, a better understanding of soil microbiome functions and processes associated with adapting agronomic management practices will be key. This is primarily due to the irreplaceable roles of the soil microbiome in soil and plant interactions, human health and ecosystem functions and stability. We approached this challenge by reviewing how different land management practices influence soil microbial communities. The important role that the soil microbiome plays in cropping systems is analyzed in the context of an overview of changes in land management over the past two decades using southern Australian dryland farming as a case study. Many major changes in management have occurred in this system during this time, including (i) reduction in tillage, with a corresponding increase in direct drilling and stubble retention; (ii) inclusion of oilseeds and pulses in cereal-dominated rotation systems; (iii) decrease in the pasture-dominated grain production systems; (iv) increased use of agrochemicals; (v) different soil amelioration applications and (vi) development of innovative agricultural technology. We review the overarching role of climate extremes in impacting agronomic management practices in southern Australian farming systems. In all, we demonstrate the complex interplay between land management changes and the activity, diversity and functions of the soil microbiome, with reference to changes in agronomic management practices.

BACKGROUND:Stomata are tiny pores on the leaf surface that are central to gas exchange. Stomatal number, size and aperture are key determinants of plant transpiration and photosynthesis, and variation in these traits can affect plant growth and productivity. Current methods to screen for stomatal phenotypes are tedious and not high throughput. This impedes research on stomatal biology and hinders efforts to develop resilient crops with optimised stomatal patterning. We have developed a rapid non-destructive method to phenotype stomatal traits in three crop species: wheat, rice and tomato.RESULTS:The method consists of two steps. The first is the non-destructive capture of images of the leaf surface from plants in their growing environment using a handheld microscope; a process that only takes a few seconds compared to minutes for other methods. The second is to analyse stomatal features using a machine learning model that automatically detects, counts and measures stomatal number, size and aperture. The accuracy of the machine learning model in detecting stomata ranged from 88 to 99%, depending on the species, with a high correlation between measures of number, size and aperture using the machine learning models and by measuring them manually. The rapid method was applied to quickly identify contrasting stomatal phenotypes.CONCLUSIONS:We developed a method that combines rapid non-destructive imaging of leaf surfaces with automated image analysis. The method provides accurate data on stomatal features while significantly reducing time for data acquisition and analysis. It can be readily used to phenotype stomata in large populations in the field and in controlled environments.

PHOSPHORUS-STARVATION TOLERANCE 1 (OsPSTOL1) is a variably present gene that benefits crown root growth and phosphorus (P) sufficiency in rice (Oryza sativa). To explore the ecophysiological importance of this gene, we performed a biogeographic survey of landraces and cultivars, confirming that functional OsPSTOL1 alleles prevail in low nutrient and drought-prone rainfed ecosystems, whereas loss-of-function and absence haplotypes predominate in control-irrigated paddy varieties of east Asia. An evolutionary history analysis of OsPSTOL1 and related genes in cereal, determined it and other genes are kinase-only domain derivatives of membrane-associated receptor like kinases. Finally, to evaluate the potential value of this kinase of unknown function in another Gramineae, wheat (Triticum aestivum) lines overexpressing OsPSTOL1 were evaluated under field and controlled low P conditions. OsPSTOL1 enhances growth, crown root number, and overall root plasticity under low P in wheat. Survey of root and shoot crown transcriptomes at two developmental stages identifies transcription factors that are differentially regulated in OsPSTOL1 wheat that are similarly controlled by the gene in rice. In wheat, OsPSTOL1 alters the timing and amplitude of regulators of root development in dry soils and hastens induction of the core P-starvation response. OsPSTOL1 and related genes may aid more sustainable cultivation of cereal crops.

Utilising a nested association mapping (NAM) population-based GWAS, 98 stable marker-trait associations with 127 alleles unique to the exotic parents were detected for grain yield and related traits in wheat. Grain yield, thousand-grain weight, screenings and hectolitre weight are important wheat yield traits. An understanding of their genetic basis is crucial for improving grain yield in breeding programmes. Nested association mapping (NAM) populations are useful resources for the dissection of the genetic basis of complex traits such as grain yield and related traits in wheat. Coupled with phenotypic data collected from multiple environments, NAM populations have the power to detect quantitative trait loci and their multiple alleles, providing germplasm that can be incorporated into breeding programmes. In this study, we evaluated a large-scale wheat NAM population with two recurrent parents in unbalanced trials in nine diverse Australian field environments over three years. By applying a single-stage factor analytical linear mixed model (FALMM) to the NAM multi-environment trials (MET) data and conducting a genome-wide association study (GWAS), we detected 98 stable marker-trait associations (MTAs) with their multiple alleles. 74 MTAs had 127 alleles that were derived from the exotic parents and were absent in either of the two recurrent parents. The exotic alleles had favourable effects on 46 MTAs of the 74 MTAs, for grain yield, thousand-grain weight, screenings and hectolitre weight. Two NAM RILs with consistently high yield in multiple environments were also identified, highlighting the potential of the NAM population in supporting plant breeding through provision of germplasm that can be readily incorporated into breeding programmes. The identified beneficial exotic alleles introgressed into the NAM population provide potential target alleles for the genetic improvement of wheat and further studies aimed at pinpointing the underlying genes.

ABSTRACT PHOSPHORUS-STARVATION TOLERANCE 1 ( OsPSTOL1 ) benefits crown root growth and phosphorus (P) sufficiency in rice ( Oryza sativa L.). To better understand the importance of this variably present gene, we carried out a biogeographic survey of landraces and cultivars, confirming that functional OsPSTOL1 alleles prevail in low nutrient and drought-prone rainfed ecosystems, whereas loss-of-function alleles and absence haplotypes predominate in control-irrigated paddy varieties of east Asia. To address the evolutionary history of OsPSTOL1 and related genes in cereal crops, phylogenetic and transcript meta-analyses were performed. Finally, to evaluate its potential value in another Gramineae, wheat ( Triticum aestivum L.) lines overexpressing OsPSTOL1 were evaluated under field and controlled low P conditions. OsPSTOL1 enhances growth, crown root number, and overall root plasticity under low P in wheat. Survey of root and shoot crown transcriptomes at two developmental stages identifies transcription factors that are differentially regulated in OsPSTOL1 wheat that are similarly controlled by the gene in rice. In wheat, OsPSTOL1 alters the timing and amplitude of regulators of root development in dry soils and hastens induction of the core P-starvation response. Based on these findings, OsPSTOL1 and related genes may aid more sustainable cultivation of cereal crops. Summary statement Might a rice gene that controls root plasticity confer a similar benefit in another grain crop. Here, we evaluate the genetic variation and evolutionary history of OsPSTOL1 and demonstrate its impact in wheat.

Research into crop yield and resilience has underpinned global food security, evident in yields tripling in the past 5 decades. The challenges that global agriculture now faces are not just to feed 10+ billion people within a generation, but to do so under a harsher, more variable, and less predictable climate, and in many cases with less water, more expensive inputs, and declining soil quality. The challenges of climate change are not simply to breed for a "hotter drier climate," but to enable resilience to floods and droughts and frosts and heat waves, possibly even within a single growing season. How well we prepare for the coming decades of climate variability will depend on our ability to modify current practices, innovate with novel breeding methods, and communicate and work with farming communities to ensure viability and profitability. Here we define how future climates will impact farming systems and growing seasons, thereby identifying the traits and practices needed and including exemplars being implemented and developed. Critically, this review will also consider societal perspectives and public engagement about emerging technologies for climate resilience, with participatory approaches presented as the best approach.

Salinity tolerance-associated phenotypes of 35 EMS mutagenized wheat lines originating from BARI Gom-25 were compared. Vegetative growth was measured using non-destructive image-based phenotyping. Five different NaCl concentrations (0 to 160 mM) were applied to plants 19 days after planting (DAP 19), and plants were imaged daily until DAP 38. Plant growth, water use, leaf Na+, K+ and Cl− content, and thousand kernel weight (TKW) were measured, and six lines were selected for further analysis. In saline conditions, leaf Na+, K+, and Cl− content variation on a dry weight basis within these six lines were ~9.3, 1.4, and 2.4-fold, respectively. Relative to BARI Gom-25, two (OA6, OA62) lines had greater K+ accumulation, three (OA6, OA10, OA62) had 50–75% lower Na+:K+ ratios, and OA62 had ~30% greater water-use index (WUI). OA23 had ~2.2-fold greater leaf Na+ and maintained TKW relative to BARI Gom-25. Two lines (OA25, OA52) had greater TKW than BARI Gom-25 when grown in 120 mM NaCl but similar Na+:K+, WUI, and biomass accumulation. OA6 had relatively high TKW, high leaf K+, and WUI, and low leaf Na+ and Cl−. Phenotypic variation revealed differing associations between the parameters measured in the lines. Future identification of the genetic basis of these differences, and crossing of lines with phenotypes of interest, is expected to enable the assessment of which combinations of parameters deliver the greatest improvement in salinity tolerance.

Salinity tolerance in bread wheat is frequently reported to be associated with low leaf sodium (Na+) concentrations. However, the Portuguese landrace, Mocho de Espiga Branca, accumulates significantly higher leaf Na+ but has comparable salinity tolerance to commercial bread wheat cultivars. To determine the genetic loci associated with the salinity tolerance of this landrace, an F2 mapping population was developed by crossing Mocho de Espiga Branca with the Australian cultivar Gladius. The population was phenotyped for 19 salinity tolerance subtraits using both non-destructive and destructive techniques. Genotyping was performed using genotyping-by-sequencing (GBS). Genomic regions associated with salinity tolerance were detected on chromosomes 1A, 1D, 4B and 5A for the subtraits of relative and absolute growth rate (RGR, AGR respectively), and on chromosome 2A, 2B, 4D and 5D for Na+, potassium (K+) and chloride (Cl-) accumulation. Candidate genes that encode proteins associated with salinity tolerance were identified within the loci including Na+/H+ antiporters, K+ channels, H+-ATPase, calcineurin B-like proteins (CBLs), CBL-interacting protein kinases (CIPKs), calcium dependent protein kinases (CDPKs) and calcium-transporting ATPase. This study provides a new insight into the genetic control of salinity tolerance in a Na+ accumulating bread wheat to assist with the future development of salt tolerant cultivars.

Proton-pumping pyrophosphatases (H+-PPases) have been shown to enhance biomass and yield. However, to date, there has been little work towards identify genes encoding H+-PPases in bread wheat (Triticum aestivum) (TaVPs) and limited knowledge on how the expression of these genes varies across different growth stages and tissue types. In this study, the IWGSC database was used to identify two novel TaVP genes, TaVP4 and TaVP5, and elucidate the complete homeolog sequences of the three known TaVP genes, bringing the total number of bread wheat TaVPs from 9 to 15. Gene expression levels of each TaVP homeolog were assessed using quantitative real-time PCR (qRT-PCR) in four diverse wheat varieties in terms of phenotypic traits related to high vacuolar pyrophosphatase expression. Homeolog expression was analyzed across multiple tissue types and developmental stages. Expression levels of the TaVP homeologs were found to vary significantly between varieties, tissues and plant developmental stages. During early development (Z10 and Z13), expressions of TaVP1 and TaVP2 homeologs were higher in shoot tissue than root tissue, with both shoot and root expression increasing in later developmental stages (Z22). TaVP2-D was expressed in all varieties and tissue types and was the most highly expressed homeolog at all developmental stages. Expression of the TaVP3 homeologs was restricted to developing grain (Z75), while TaVP4 homeolog expression was higher at Z22 than earlier developmental stages. Variation in TaVP4B was detected among varieties at Z22 and Z75, with Buck Atlantico (high biomass) and Scout (elite Australian cultivar) having the highest levels of expression. These findings offer a comprehensive overview of the bread wheat H+-PPase family and identify variation in TaVP homeolog expression that will be of use to improve the growth, yield, and abiotic stress tolerance of bread wheat.

Improving salinity tolerance in the most widely cultivated cereal, bread wheat (Triticum aestivum L.), is essential to increase grain yields on saline agricultural lands. A Portuguese landrace, Mocho de Espiga Branca accumulates up to sixfold greater leaf and sheath sodium (Na+ ) than two Australian cultivars, Gladius and Scout, under salt stress in hydroponics. Despite high leaf and sheath Na+ concentrations, Mocho de Espiga Branca maintained similar salinity tolerance compared to Gladius and Scout. A naturally occurring single nucleotide substitution was identified in the gene encoding a major Na+ transporter TaHKT1;5-D in Mocho de Espiga Branca, which resulted in a L190P amino acid residue variation. This variant prevents Mocho de Espiga Branca from retrieving Na+ from the root xylem leading to a high shoot Na+ concentration. The identification of the tissue-tolerant Mocho de Espiga Branca will accelerate the development of more elite salt-tolerant bread wheat cultivars.

ABSTRACT We quantified grain sodium (Na + ) content across a barley GWAS panel grown under optimal conditions. We identified a strong association with a region containing two low and one high Na + accumulating haplotypes of a Class 1 HIGH-AFFINITY POTASSIUM TRANSPORTER (HKT1;5) known to be involved in regulating plant Na + homeostasis. The haplotypes exhibited an average 1.8-fold difference in grain Na + content. We show that an L189P substitution disrupts Na + transport in the high Na + lines, disturbs the plasma membrane localisation typical of HKT1;5 and induces a conformational change in the protein predicted to compromise function. Under NaCl stress, lines containing P189 accumulate high levels of Na + , but show no significant difference in biomass. P189 increases in frequency from wild-species to elite cultivars leading us to speculate that the compromised haplotype is undergoing directional selection possibly due to the value of Na + as a functional nutrient in non-saline environments.

During plant growth, sodium (Na + ) in the soil is transported via the xylem from the root to the shoot. While excess Na + is toxic to most plants, non-toxic concentrations have been shown to improve crop yields under certain conditions, such as when soil K + is low. We quantified grain Na + across a barley genome-wide association study panel grown under non-saline conditions and identified variants of a Class 1 HIGH-AFFINITY-POTASSIUM-TRANSPORTER ( HvHKT1;5 )-encoding gene responsible for Na + content variation under these conditions. A leucine to proline substitution at position 189 (L189P) in HvHKT1;5 disturbs its characteristic plasma membrane localisation and disrupts Na + transport. Under low and moderate soil Na + , genotypes containing HvHKT1:5 P189 accumulate high concentrations of Na + but exhibit no evidence of toxicity. As the frequency of HvHKT1:5 P189 increases significantly in cultivated European germplasm, we cautiously speculate that this non-functional variant may enhance yield potential in non-saline environments, possibly by offsetting limitations of low available K + .

A fundamental factor to improve crop productivity involves the optimization of reduced carbon translocation from source to sink tissues. Here, we present data consistent with the positive effect that the expression of the Arabidopsis thaliana H+-PPase (AVP1) has on reduced carbon partitioning and yield increases in wheat. Immunohistochemical localization of H+-PPases (TaVP) in spring wheat Bobwhite L. revealed the presence of this conserved enzyme in wheat vasculature and sink tissues. Of note, immunogold imaging showed a plasma membrane localization of TaVP in sieve element- companion cell complexes of Bobwhite source leaves. These data together with the distribution patterns of a fluorescent tracer and [U14C]-sucrose are consistent with an apoplasmic phloem-loading model in wheat. Interestingly, 14C-labeling experiments provided evidence for enhanced carbon partitioning between shoots and roots, and between flag leaves and milk stage kernels in AVP1 expressing Bobwhite lines. In keeping, there is a significant yield improvement triggered by the expression of AVP1 in these lines. Green house and field grown transgenic wheat expressing AVP1 also produced higher grain yield and number of seeds per plant, and exhibited an increase in root biomass when compared to null segregants. Another agriculturally desirable phenotype showed by AVP1 Bobwhite plants is a robust establishment of seedlings.