Potential abiotic stress targets for modern genetic manipulation.

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.

Diversity in bread and durum wheat stigma morphology and linkage of increased stigma length to dwarfing gene Rht14.

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.

PHOSPHORUS-STARVATION TOLERANCE 1 (OsPSTOL1) is prevalent in upland rice and enhances root growth and hastens low phosphate signaling in wheat.

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.

Nested association mapping-based GWAS for grain yield and related traits in wheat grown under diverse Australian environments.

KEY MESSAGE: 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.

Improved Salinity Tolerance-Associated Variables Observed in EMS Mutagenized Wheat Lines.

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.

High-throughput 3D modelling to dissect the genetic control of leaf elongation in barley (Hordeum vulgare).

To optimize shoot growth and structure of cereals, we need to understand the genetic components controlling initiation and elongation. While measuring total shoot growth at high throughput using 2D imaging has progressed, recovering the 3D shoot structure of small grain cereals at a large scale is still challenging. Here, we present a method for measuring defined individual leaves of cereals, such as wheat and barley, using few images. Plant shoot modelling over time was used to measure the initiation and elongation of leaves in a bi-parental barley mapping population under low and high soil salinity. We detected quantitative trait loci (QTL) related to shoot growth per se, using both simple 2D total shoot measurements and our approach of measuring individual leaves. In addition, we detected QTL specific to leaf elongation and not to total shoot size. Of particular importance was the detection of a QTL on chromosome 3H specific to the early responses of leaf elongation to salt stress, a locus that could not be detected without the computer vision tools developed in this study.

Energy costs of salt tolerance in crop plants.

Agriculture is expanding into regions that are affected by salinity. This review considers the energetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion uptake are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H+ -ATPase also is a critical component. One proposed leak, that of Na+ influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na+ and Cl- concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will inform experimentation and allow a quantitative assessment of the energy costs of NaCl tolerance to guide breeding and engineering of molecular components.

A single nucleotide substitution in TaHKT1;5-D controls shoot Na+ accumulation in 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.

The structural validity of the IKDC and its relationship with quality of life following ACL reconstruction.

OBJECTIVE: The purpose of this study was to (a) examine the structural validity of the International Knee Documentation Committee Subjective Knee Form in light of previously reported dimensionality issues, and (b) examine the relationships between the IKDC and patients' knee-related quality of life 2-9 years after anterior cruciate ligament (ACL) reconstruction. METHODS: A prospective research design was employed, wherein 319 patients (mean age = 29.07, SD = 9.03) completed the IKDC before surgery, 191 patients (mean age = 29.71, SD = 9.36) completed the IKDC at 6 months post-surgery, and 132 patients (mean age = 34.34, SD = 7.89) completed the IKDC and the Anterior Cruciate Ligament Quality of Life Survey (ACL-QOL) at 2-9 years post-surgery. RESULTS: Bayesian structural equation modeling analysis confirmed the two-factor structure (symptom & knee articulation and activity level) represented the most accurate conceptualization of perceived knee function across the three time-points. Moreover, findings revealed that of the two IKDC subscales pre-operatively, activity level was most strongly associated with long-term quality of life at 2-9 years following surgery, whereas 2-9 years post-operatively, symptoms and knee articulation was most strongly associated with long-term quality of life. CONCLUSIONS: The IKDC provides clinicians with a convenient total score to assess patients' perceived knee function, but its unidimensional factor structure is a poor representation of its items and fails to detect discrepancies in patients' post-operative quality of life, such as the relative importance of perceived knee activity level before reconstructive surgery.

Translating knowledge about abiotic stress tolerance to breeding programmes.

Plant breeding and improvements in agronomic practice are making a consistent contribution to increasing global crop production year upon year. However, the rate of yield improvement currently lags behind the targets set to produce enough food to meet the demands of the predicted global population in 2050. Furthermore, crops that are exposed to harmful abiotic environmental factors (abiotic stresses, e.g. water limitation, salinity, extreme temperature) are prone to reduced yields. Here, we briefly describe the processes undertaken in conventional breeding programmes, which are usually designed to improve yields in near-optimal conditions rather than specifically breeding for improved crop yield stability under stressed conditions. While there is extensive fundamental research activity that examines mechanisms of plant stress tolerance, there are few examples that apply this research to improving commercial crop yields. There are notable exceptions, and we highlight some of these to demonstrate the magnitude of yield gains that could be made by translating agronomic, phenological and genetic solutions focused on improving or mitigating the effect of abiotic stress in the field; in particular, we focus on improvements in crop water-use efficiency and salinity tolerance. We speculate upon the reasons for the disconnect between research and research translation. We conclude that to realise untapped rapid gains towards food security targets new funding structures need to be embraced. Such funding needs to serve both the core and collaborative activities of the fundamental, pre-breeding and breeding research communities in order to expedite the translation of innovative research into the fields of primary producers.

Mapping of novel salt tolerance QTL in an Excalibur × Kukri doubled haploid wheat population.

KEY MESSAGE: Novel QTL for salinity tolerance traits have been detected using non-destructive and destructive phenotyping in bread wheat and were shown to be linked to improvements in yield in saline fields. Soil salinity is a major limitation to cereal production. Breeding new salt-tolerant cultivars has the potential to improve cereal crop yields. In this study, a doubled haploid bread wheat mapping population, derived from the bi-parental cross of Excalibur × Kukri, was grown in a glasshouse under control and salinity treatments and evaluated using high-throughput non-destructive imaging technology. Quantitative trait locus (QTL) analysis of this population detected multiple QTL under salt and control treatments. Of these, six QTL were detected in the salt treatment including one for maintenance of shoot growth under salinity (QG(1-5).asl-7A), one for leaf Na+ exclusion (QNa.asl-7A) and four for leaf K+ accumulation (QK.asl-2B.1, QK.asl-2B.2, QK.asl-5A and QK:Na.asl-6A). The beneficial allele for QG(1-5).asl-7A (the maintenance of shoot growth under salinity) was present in six out of 44 mainly Australian bread and durum wheat cultivars. The effect of each QTL allele on grain yield was tested in a range of salinity concentrations at three field sites across 2 years. In six out of nine field trials with different levels of salinity stress, lines with alleles for Na+ exclusion and/or K+ maintenance at three QTL (QNa.asl-7A, QK.asl-2B.2 and QK:Na.asl-6A) excluded more Na+ or accumulated more K+ compared to lines without these alleles. Importantly, the QK.asl-2B.2 allele for higher K+ accumulation was found to be associated with higher grain yield at all field sites. Several alleles at other QTL were associated with higher grain yields at selected field sites.

Genetics of Na+ exclusion and salinity tolerance in Afghani durum wheat landraces.

BACKGROUND: Selecting for low concentration of Na+ in the shoot provides one approach for tackling salinity stress that adversely affects crop production. Novel alleles for Na+ exclusion can be identified and then introduced into elite crop cultivars. RESULTS: We have identified loci associated with lower Na+ concentration in leaves of durum wheat landraces originating from Afghanistan. Seedlings of two F2 populations derived from crossings between Australian durum wheat (Jandaroi) and two Afghani landraces (AUS-14740 and AUS-14752) were grown hydroponically and evaluated for Na+ and K+ concentration in the third leaf. High heritability was found for both third leaf Na+ concentration and the K+/Na+ ratio in both populations. Further work focussed on line AUS-14740. Bulk segregant analysis using 9 K SNP markers identified two loci significantly associated with third leaf Na+ concentration. Marker regression analysis showed a strong association between all traits studied and a favourable allele originating from AUS-14740 located on the long arm of chromosome 4B. CONCLUSIONS: The candidate gene in the relevant region of chromosome 4B is likely to be the high affinity K+ transporter B1 (HKT1;5-B1). A second locus associated with third leaf Na+ concentration was located on chromosome 3BL, with the favourable allele originating from Jandaroi; however, no candidate gene can be identified.

Identification of a Stelar-Localized Transport Protein That Facilitates Root-to-Shoot Transfer of Chloride in Arabidopsis.

Under saline conditions, higher plants restrict the accumulation of chloride ions (Cl(-)) in the shoot by regulating their transfer from the root symplast into the xylem-associated apoplast. To identify molecular mechanisms underpinning this phenomenon, we undertook a transcriptional screen of salt stressed Arabidopsis (Arabidopsis thaliana) roots. Microarrays, quantitative RT-PCR, and promoter-GUS fusions identified a candidate gene involved in Cl(-) xylem loading from the Nitrate transporter 1/Peptide Transporter family (NPF2.4). This gene was highly expressed in the root stele compared to the cortex, and its expression decreased after exposure to NaCl or abscisic acid. NPF2.4 fused to fluorescent proteins, expressed either transiently or stably, was targeted to the plasma membrane. Electrophysiological analysis of NPF2.4 in Xenopus laevis oocytes suggested that NPF2.4 catalyzed passive Cl(-) efflux out of cells and was much less permeable to NO3(-). Shoot Cl(-) accumulation was decreased following NPF2.4 artificial microRNA knockdown, whereas it was increased by overexpression of NPF2.4. Taken together, these results suggest that NPF2.4 is involved in long-distance transport of Cl(-) in plants, playing a role in the loading and the regulation of Cl(-) loading into the xylem of Arabidopsis roots during salinity stress.

The evolutionary origin of CIPK16: A gene involved in enhanced salt tolerance.

Calcineurin B-like protein interacting protein kinases (CIPKs) are key regulators of pre-transcriptional and post-translational responses to abiotic stress. Arabidopsis thaliana CIPK16 (AtCIPK16) was identified from a forward genetic screen as a gene that mediates lower shoot salt accumulation and improved salinity tolerance in Arabidopsis and transgenic barley. Here, we aimed to gain an understanding of the evolution of AtCIPK16, and orthologues of CIPK16 in other plant species including barley, by conducting a phylogenetic analysis of terrestrial plant species. The resulting protein sequence based phylogenetic trees revealed a single clade that included AtCIPK16 along with two segmentally duplicated CIPKs, AtCIPK5 and AtCIPK25. No monocots had proteins that fell into this clade; instead the most closely related monocot proteins formed a group basal to the entire CIPK16, 5 and 25 clade. We also found that AtCIPK16 contains a core Brassicales specific indel and a putative nuclear localisation signal, which are synapomorphic characters of CIPK16 genes. In addition, we present a model that proposes the evolution of CIPK16, 5 and 25 clade.

SLAH1, a homologue of the slow type anion channel SLAC1, modulates shoot Cl- accumulation and salt tolerance in Arabidopsis thaliana.

Salinity tolerance is correlated with shoot chloride (Cl(-)) exclusion in multiple crops, but the molecular mechanisms of long-distance Cl(-) transport are poorly defined. Here, we characterize the in planta role of AtSLAH1 (a homologue of the slow type anion channel-associated 1 (SLAC1)). This protein, localized to the plasma membrane of root stelar cells, has its expression reduced by salt or ABA, which are key predictions for a protein involved with loading Cl(-) into the root xylem. Artificial microRNA knockdown mutants of AtSLAH1 had significantly reduced shoot Cl(-) accumulation when grown under low Cl(-), whereas shoot Cl(-) increased and the shoot nitrate/chloride ratio decreased following AtSLAH1 constitutive or stelar-specific overexpression when grown in high Cl(-) In both sets of overexpression lines a significant reduction in shoot biomass over the null segregants was observed under high Cl(-) supply, but not low Cl(-) supply. Further in planta data showed AtSLAH3 overexpression increased the shoot nitrate/chloride ratio, consistent with AtSLAH3 favouring nitrate transport. Heterologous expression of AtSLAH1 in Xenopus laevis oocytes led to no detectible transport, suggesting the need for post-translational modifications for AtSLAH1 to be active. Our in planta data are consistent with AtSLAH1 having a role in controlling root-to-shoot Cl(-) transport.

Evaluating contribution of ionic, osmotic and oxidative stress components towards salinity tolerance in barley.

BACKGROUND: Salinity tolerance is a physiologically multi-faceted trait attributed to multiple mechanisms. Three barley (Hordeum vulgare) varieties contrasting in their salinity tolerance were used to assess the relative contribution of ionic, osmotic and oxidative stress components towards overall salinity stress tolerance in this species, both at the whole-plant and cellular levels. In addition, transcriptional changes in the gene expression profile were studied for key genes mediating plant ionic and oxidative homeostasis (NHX; RBOH; SOD; AHA and GORK), to compare a contribution of transcriptional and post-translational factors towards the specific components of salinity tolerance. RESULTS: Our major findings are two-fold. First, plant tissue tolerance was a dominating component that has determined the overall plant responses to salinity, with root K(+) retention ability and reduced sensitivity to stress-induced hydroxyl radical production being the main contributing tolerance mechanisms. Second, it was not possible to infer which cultivars were salinity tolerant based solely on expression profiling of candidate genes at one specific time point. For the genes studied and the time point selected that transcriptional changes in the expression of these specific genes had a small role for barley's adaptive responses to salinity. CONCLUSIONS: For better tissue tolerance, sodium sequestration, K(+) retention and resistance to oxidative stress all appeared to be crucial. Because these traits are highly interrelated, it is suggested that a major progress in crop breeding for salinity tolerance can be achieved only if these complementary traits are targeted at the same time. This study also highlights the essentiality of post translational modifications in plant adaptive responses to salinity.

Expression of the Arabidopsis vacuolar H⁺-pyrophosphatase gene (AVP1) improves the shoot biomass of transgenic barley and increases grain yield in a saline field.

Cereal varieties with improved salinity tolerance are needed to achieve profitable grain yields in saline soils. The expression of AVP1, an Arabidopsis gene encoding a vacuolar proton pumping pyrophosphatase (H⁺-PPase), has been shown to improve the salinity tolerance of transgenic plants in greenhouse conditions. However, the potential for this gene to improve the grain yield of cereal crops in a saline field has yet to be evaluated. Recent advances in high-throughput nondestructive phenotyping technologies also offer an opportunity to quantitatively evaluate the growth of transgenic plants under abiotic stress through time. In this study, the growth of transgenic barley expressing AVP1 was evaluated under saline conditions in a pot experiment using nondestructive plant imaging and in a saline field trial. Greenhouse-grown transgenic barley expressing AVP1 produced a larger shoot biomass compared to null segregants, as determined by an increase in projected shoot area, when grown in soil with 150 mM NaCl. This increase in shoot biomass of transgenic AVP1 barley occurred from an early growth stage and also in nonsaline conditions. In a saline field, the transgenic barley expressing AVP1 also showed an increase in shoot biomass and, importantly, produced a greater grain yield per plant compared to wild-type plants. Interestingly, the expression of AVP1 did not alter barley leaf sodium concentrations in either greenhouse- or field-grown plants. This study validates our greenhouse-based experiments and indicates that transgenic barley expressing AVP1 is a promising option for increasing cereal crop productivity in saline fields.

HVP10 encoding V-PPase is a prime candidate for the barley HvNax3 sodium exclusion gene: evidence from fine mapping and expression analysis.

In cereals, a common salinity tolerance mechanism is to limit accumulation of Na(+) in the shoot. In a cross between the barley variety Barque-73 (Hordeum vulgare ssp. vulgare) and the accession CPI-71284 of wild barley (H. vulgare ssp. spontaneum), the HvNax3 locus on chromosome 7H was found to determine a ~10-25 % difference in leaf Na(+) accumulation in seedlings grown in saline hydroponics, with the beneficial exclusion trait originating from the wild parent. The Na(+) exclusion allele was also associated with a 13-21 % increase in shoot fresh weight. The HvNax3 locus was delimited to a 0.4 cM genetic interval, where it cosegregated with the HVP10 gene for vacuolar H(+)-pyrophosphatase (V-PPase). Sequencing revealed that the mapping parents encoded identical HVP10 proteins, but salinity-induced mRNA expression of HVP10 was higher in CPI-71284 than in Barque-73, in both roots and shoots. By contrast, the expression of several other genes predicted by comparative mapping to be located in the HvNax3 interval was similar in the two parent lines. Previous work demonstrated roles for V-PPase in ion transport and salinity tolerance. We therefore considered transcription levels of HVP10 to be a possible basis for variation in shoot Na(+) accumulation and biomass production controlled by the HvNax3 locus under saline conditions. Potential mechanisms linking HVP10 expression patterns to the observed phenotypes are discussed.

Rapid non-destructive method to phenotype stomatal traits.

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.