Water Stress Alters Physiological, Spectral, and Agronomic Indexes of Wheat Genotypes.

Selecting drought-tolerant and more water-efficient wheat genotypes is a research priority, specifically in regions with irregular rainfall or areas where climate change is expected to result in reduced water availability. The objective of this work was to use high-throughput measurements with morphophysiological traits to characterize wheat genotypes in relation to water stress. Field experiments were conducted from May to September 2018 and 2019, using a sprinkler bar irrigation system to control water availability to eighteen wheat genotypes: BRS 254; BRS 264; CPAC 01019; CPAC 01047; CPAC 07258; CPAC 08318; CPAC 9110; BRS 394 (irrigated biotypes), and Aliança; BR 18_Terena; BRS 404; MGS Brilhante; PF 020037; PF 020062; PF 120337; PF 100368; PF 080492; and TBIO Sintonia (rainfed biotypes). The water regimes varied from 22 to 100% of the crop evapotranspiration replacement. Water stress negatively affected gas exchange, vegetation indices, and grain yield. High throughput variables TCARI, NDVI, OSAVI, SAVI, PRI, NDRE, and GNDVI had higher yield and morphophysiological measurement correlations. The drought resistance index indicated that genotypes Aliança, BRS 254, BRS 404, CPAC 01019, PF 020062, and PF 080492 were more drought tolerant.

Molecular Interplay between Non-Host Resistance, Pathogens and Basal Immunity as a Background for Fatal Yellowing in Oil Palm (Elaeis guineensis Jacq.) Plants.

An oil palm (Elaeis guineensis Jacq.) bud rod disorder of unknown etiology, named Fatal Yellowing (FY) disease, is regarded as one of the top constraints with respect to the growth of the palm oil industry in Brazil. FY etiology has been a challenge embraced by several research groups in plant pathology throughout the last 50 years in Brazil, with no success in completing Koch's postulates. Most recently, the hypothesis of having an abiotic stressor as the initial cause of FY has gained ground, and oxygen deficiency (hypoxia) damaging the root system has become a candidate for stress. Here, a comprehensive, large-scale, single- and multi-omics integration analysis of the metabolome and transcriptome profiles on the leaves of oil palm plants contrasting in terms of FY symptomatology-asymptomatic and symptomatic-and collected in two distinct seasons-dry and rainy-is reported. The changes observed in the physicochemical attributes of the soil and the chemical attributes and metabolome profiles of the leaves did not allow the discrimination of plants which were asymptomatic or symptomatic for this disease, not even in the rainy season, when the soil became waterlogged. However, the multi-omics integration analysis of enzymes and metabolites differentially expressed in asymptomatic and/or symptomatic plants in the rainy season compared to the dry season allowed the identification of the metabolic pathways most affected by the changes in the environment, opening an opportunity for additional characterization of the role of hypoxia in FY symptom intensification. Finally, the initial analysis of a set of 56 proteins/genes differentially expressed in symptomatic plants compared to the asymptomatic ones, independent of the season, has presented pieces of evidence suggesting that breaks in the non-host resistance to non-adapted pathogens and the basal immunity to adapted pathogens, caused by the anaerobic conditions experienced by the plants, might be linked to the onset of this disease. This set of genes might offer the opportunity to develop biomarkers for selecting oil palm plants resistant to this disease and to help pave the way to employing strategies to keep the safety barriers raised and strong.

Osmoprotectants play a major role in the Portulaca oleracea resistance to high levels of salinity stress-insights from a metabolomics and proteomics integrated approach.

Introduction: Purslane (Portulaca oleracea L.) is a non-conventional food plant used extensively in folk medicine and classified as a multipurpose plant species, serving as a source of features of direct importance to the agricultural and agri-industrial sectors. This species is considered a suitable model to study the mechanisms behind resistance to several abiotic stresses including salinity. The recently achieved technological developments in high-throughput biology opened a new window of opportunity to gain additional insights on purslane resistance to salinity stress-a complex, multigenic, and still not well-understood trait. Only a few reports on single-omics analysis (SOA) of purslane are available, and only one multi-omics integration (MOI) analysis exists so far integrating distinct omics platforms (transcriptomics and metabolomics) to characterize the response of purslane plants to salinity stress. Methods: The present study is a second step in building a robust database on the morpho-physiological and molecular responses purslane to salinity stress and its subsequent use in attempting to decode the genetics behind its resistance to this abiotic stress. Here, the characterization of the morpho-physiological responses of adult purslane plants to salinity stress and a metabolomics and proteomics integrative approach to study the changes at the molecular level in their leaves and roots is presented. Results and discussion: Adult plants of the B1 purslane accession lost approximately 50% of the fresh and dry weight (from shoots and roots) whensubmitted to very high salinity stress (2.0 g of NaCl/100 g of the substrate). The resistance to very high levels of salinity stress increases as the purslane plant matures, and most of the absorbed sodium remains in the roots, with only a part (~12%) reaching the shoots. Crystal-like structures, constituted mainly by Na+, Cl-, and K+, were found in the leaf veins and intercellular space near the stoma, indicating that this species has a mechanism of salt exclusion operating on the leaves, which has its role in salt tolerance. The MOI approach showed that 41 metabolites were statistically significant on the leaves and 65 metabolites on the roots of adult purslane plants. The combination of the mummichog algorithm and metabolomics database comparison revealed that the glycine, serine, and threonine, amino sugar and nucleotide sugar, and glycolysis/gluconeogenesis pathways were the most significantly enriched pathways when considering the total number of occurrences in the leaves (with 14, 13, and 13, respectively) and roots (all with eight) of adult plants; and that purslane plants employ the adaptive mechanism of osmoprotection to mitigate the negative effect of very high levels of salinity stress; and that this mechanism is prevalent in the leaves. The multi-omics database built by our group underwent a screen for salt-responsive genes, which are now under further characterization for their potential to promote resistance to salinity stress when heterologously overexpressed in salt-sensitive plants.

Use of Thermography to Evaluate Alternative Crops for Off-Season in the Cerrado Region.

Future predictions due to climate change are of decreases in rainfall and longer drought periods. The search for new tolerant crops is an important strategy. The objective of this study was to evaluate the effect of water stress on the physiology and productivity of crops with potential for growing in the off-season period in the Cerrado, and evaluate correlations with the temperature of the canopy obtained by means of thermography. The experiment was conducted under field conditions, with experimental design in randomized blocks, in a split-plot scheme and four replications. The plots were: common bean (Phaseolus vulgaris); amaranth (Amaranthus cruentus); quinoa (Chenopodium quinoa); and buckwheat (Fagopyrum esculentum). The subplots were composed of four water regimes: maximum water regime (WR 535 mm), high-availability regime (WR 410 mm), off-season water regime (WR 304 mm) and severe water regime (WR 187 mm). Under WR 304 mm, the internal concentration of CO2 and photosynthesis were reduced by less than 10% in amaranth. Common bean and buckwheat reduced 85% in photosynthesis. The reduction in water availability increased the canopy temperature in the four crops and, in general, common bean was the most sensitive species, while quinoa had the lowest canopy temperatures. Furthermore, canopy temperature correlated negatively with grain yield, biomass yield and gas exchange across all plant species, thus thermal imaging of the canopy represents a promising tool for monitoring crop productivity for farmers, For the identification of crops with high water use management for research.

Multi-omics Analysis of Young Portulaca oleracea L. Plants' Responses to High NaCl Doses Reveals Insights into Pathways and Genes Responsive to Salinity Stress in this Halophyte Species.

Soil salinity is among the abiotic stressors that threaten agriculture the most, and purslane (Portulaca oleracea L.) is a dicot species adapted to inland salt desert and saline habitats that hyper accumulates salt and has high phytoremediation potential. Many researchers consider purslane a suitable model species to study the mechanisms of plant tolerance to drought and salt stresses. Here, a robust salinity stress protocol was developed and used to characterize the morphophysiological responses of young purslane plants to salinity stress; then, leaf tissue underwent characterization by distinct omics platforms to gain further insights into its response to very high salinity stress. The salinity stress protocol did generate different levels of stress by gradients of electrical conductivity at field capacity and water potential in the saturation extract of the substrate, and the morphological parameters indicated three distinct stress levels. As expected from a halophyte species, these plants remained alive under very high levels of salinity stress, showing salt crystal-like structures constituted mainly by Na+, Cl-, and K+ on and around closed stomata. A comprehensive and large-scale metabolome and transcriptome single and integrated analyses were then employed using leaf samples. The multi-omics integration (MOI) system analysis led to a data-set of 51 metabolic pathways with at least one enzyme and one metabolite differentially expressed due to salinity stress. These data sets (of genes and metabolites) are valuable for future studies aimed to deepen our knowledge on the mechanisms behind the high tolerance of this species to salinity stress. In conclusion, besides showing that this species applies salt exclusion already in young plants to support very high levels of salinity stress, the initial analysis of metabolites and transcripts data sets already give some insights into other salt tolerance mechanisms used by this species to support high levels of salinity stress. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-022-00061-2.

Structural and functional analysis of stress-inducible genes and their promoters selected from young oil palm (Elaeis guineensis) under salt stress.

BACKGROUND: Soil salinity is a problem in more than 100 countries across all continents. It is one of the abiotic stress that threatens agriculture the most, negatively affecting crops and reducing productivity. Transcriptomics is a technology applied to characterize the transcriptome in a cell, tissue, or organism at a given time via RNA-Seq, also known as full-transcriptome shotgun sequencing. This technology allows the identification of most genes expressed at a particular stage, and different isoforms are separated and transcript expression levels measured. Once determined by this technology, the expression profile of a gene must undergo validation by another, such as quantitative real-time PCR (qRT-PCR). This study aimed to select, annotate, and validate stress-inducible genes-and their promoters-differentially expressed in the leaves of oil palm (Elaeis guineensis) plants under saline stress. RESULTS: The transcriptome analysis led to the selection of 14 genes that underwent structural and functional annotation, besides having their expression validated using the qRT-PCR technique. When compared, the RNA-Seq and qRT-PCR profiles of those genes resulted in some inconsistencies. The structural and functional annotation analysis of proteins coded by the selected genes showed that some of them are orthologs of genes reported as conferring resistance to salinity in other species. There were those coding for proteins related to the transport of salt into and out of cells, transcriptional regulatory activity, and opening and closing of stomata. The annotation analysis performed on the promoter sequence revealed 22 distinct types of cis-acting elements, and 14 of them are known to be involved in abiotic stress. CONCLUSION: This study has helped validate the process of an accurate selection of genes responsive to salt stress with a specific and predefined expression profile and their promoter sequence. Its results also can be used in molecular-genetics-assisted breeding programs. In addition, using the identified genes is a window of opportunity for strategies trying to relieve the damages arising from the salt stress in many glycophyte crops with economic importance.

The early response of oil palm (Elaeis guineensis Jacq.) plants to water deprivation: Expression analysis of miRNAs and their putative target genes, and similarities with the response to salinity stress.

Oil palm (Elaeis guineensis Jacq.) is a oilseed crop of great economic importance drastically affected by abiotic stresses. MicroRNAs (miRNAs) play crucial roles in transcription and post-transcription regulation of gene expression, being essential molecules in the response of plants to abiotic stress. To better understand the molecular mechanisms behind the response of young oil palm plants to drought stress, this study reports on the prediction and characterization of miRNAs and their putative target genes in the apical leaf of plants subjected to 14 days of water deprivation. Then, the data from this study were compared to the data from a similar study that focused on salinity stress. Both, the drought-and salt-responsive miRNAs and their putative target genes underwent correlation analysis to identify similarities and dissimilarities among them. Among the 81 identified miRNAs, 29 are specific for oil palm, including two (egu-miR28ds and egu-miR29ds) new ones - described for the first time. As for the expression profile, 62 miRNAs were significantly differentially expressed under drought stress, being five up-regulated (miR396e, miR159b, miR529b, egu-miR19sds, and egu-miR29ds) and 57 down-regulated. Transcription factors, such as MYBs, HOXs, and NF-Ys, were predicted as putative miRNA-target genes in oil palm under water deprivation; making them the most predominant group of such genes. Finally, the correlation analysis study revealed a group of putative target genes with similar behavior under salt and drought stresses. Those genes that are upregulated by these two abiotic stresses encode lncRNAs and proteins linked to stress tolerance, stress memory, modulation of ROS signaling, and defense response regulation to abiotic and biotic stresses. In summary, this study provides molecular evidence for the possible involvement of miRNAs in the drought stress response in oil palm. Besides, it shows that, at the molecular level, there are many similarities in the response of young oil palm plants to these two abiotic stresses.

Insights from a Multi-Omics Integration (MOI) Study in Oil Palm (Elaeis guineensis Jacq.) Response to Abiotic Stresses: Part Two-Drought.

Drought and salinity are two of the most severe abiotic stresses affecting agriculture worldwide and bear some similarities regarding the responses of plants to them. The first is also known as osmotic stress and shows similarities mainly with the osmotic effect, the first phase of salinity stress. Multi-Omics Integration (MOI) offers a new opportunity for the non-trivial challenge of unraveling the mechanisms behind multigenic traits, such as drought and salinity resistance. The current study carried out a comprehensive, large-scale, single-omics analysis (SOA) and MOI studies on the leaves of young oil palm plants submitted to water deprivation. After performing SOA, 1955 DE enzymes from transcriptomics analysis, 131 DE enzymes from proteomics analysis, and 269 DE metabolites underwent MOI analysis, revealing several pathways affected by this stress, with at least one DE molecule in all three omics platforms used. Moreover, the similarities and dissimilarities in the molecular response of those plants to those two abiotic stresses underwent mapping. Cysteine and methionine metabolism (map00270) was the most affected pathway in all scenarios evaluated. The correlation analysis revealed that 91.55% of those enzymes expressed under both stresses had similar qualitative profiles, corroborating the already known fact that plant responses to drought and salinity show several similarities. At last, the results shed light on some candidate genes for engineering crop species resilient to both abiotic stresses.

Insights from a Multi-Omics Integration (MOI) Study in Oil Palm (Elaeis guineensis Jacq.) Response to Abiotic Stresses: Part One-Salinity.

Oil palm (Elaeis guineensis Jacq.) is the number one source of consumed vegetable oil nowadays. It is cultivated in areas of tropical rainforest, where it meets its natural condition of high rainfall throughout the year. The palm oil industry faces criticism due to a series of practices that was considered not environmentally sustainable, and it finds itself under pressure to adopt new and innovative procedures to reverse this negative public perception. Cultivating this oilseed crop outside the rainforest zone is only possible using artificial irrigation. Close to 30% of the world's irrigated agricultural lands also face problems due to salinity stress. Consequently, the research community must consider drought and salinity together when studying to empower breeding programs in order to develop superior genotypes adapted to those potential new areas for oil palm cultivation. Multi-Omics Integration (MOI) offers a new window of opportunity for the non-trivial challenge of unraveling the mechanisms behind multigenic traits, such as drought and salinity tolerance. The current study carried out a comprehensive, large-scale, single-omics analysis (SOA), and MOI study on the leaves of young oil palm plants submitted to very high salinity stress. Taken together, a total of 1239 proteins were positively regulated, and 1660 were negatively regulated in transcriptomics and proteomics analyses. Meanwhile, the metabolomics analysis revealed 37 metabolites that were upregulated and 92 that were downregulated. After performing SOA, 436 differentially expressed (DE) full-length transcripts, 74 DE proteins, and 19 DE metabolites underwent MOI analysis, revealing several pathways affected by this stress, with at least one DE molecule in all three omics platforms used. The Cysteine and methionine metabolism (map00270) and Glycolysis/Gluconeogenesis (map00010) pathways were the most affected ones, each one with 20 DE molecules.

Deep Untargeted Metabolomics Analysis to Further Characterize the Adaptation Response of Gliricidia sepium (Jacq.) Walp. to Very High Salinity Stress.

The multipurpose tree Gliricidia sepium (Jacq.) Walp. adapts to a very high level of salt stress (≥20 dS m-1) and resumes the production of new leaves around 2 weeks after losing all leaves due to abrupt salinity stress. The integration of metabolome and transcriptome profiles from gliricidia leaves points to a central role of the phenylpropanoid biosynthesis pathway in the short-term response to salinity stress. In this study, a deeper untargeted metabolomics analysis of the leaves and roots of young gliricidia plants was conducted to characterize the mechanism(s) behind this adaptation response. The polar and lipidic fractions from leaf and root samples were extracted and analyzed on a UHPLC.ESI.Q-TOF.HRMS system. Acquired data were analyzed using the XCMS Online, and MetaboAnalyst platforms, via three distinct and complementary strategies. Together, the results obtained first led us to postulate that these plants are salt-excluding plants, which adapted to high salinity stress via two salt-excluding mechanisms, starting in the canopy-severe defoliation-and concluding in the roots-limited entry of Na. Besides that, it was possible to show that the phenylpropanoid biosynthesis pathway plays a role throughout the entire adaptation response, starting in the short term and continuing in the long one. The roots metabolome analysis revealed 11 distinct metabolic pathways affected by salt stress, and the initial analysis of the two most affected ones-steroid biosynthesis and lysine biosynthesis-led us also to postulate that the accumulation of lignin and some phytosterols, as well as lysine biosynthesis-but not degradation, play a role in promoting the adaptation response. However, additional studies are necessary to investigate these hypotheses.

Water Stress Alters Morphophysiological, Grain Quality and Vegetation Indices of Soybean Cultivars.

Rainfall is among the climatic factors that most affect production, as in the Brazilian Cerrado. Non-destructive and automated phenotyping methods are fast and efficient for genotype selection. The objective of this work was to evaluate, under field conditions, the morphophysiological changes, yield, and grain quality of soybean (Glycine max L. Merrill) under water stress in the Brazilian Cerrado. The plots comprised six soybean cultivars and the subplots of four water regimes, corresponding to 31, 44, 64 and 100% of crop evapotranspiration replacement. The experiments were conducted from May to September 2018 and 2019. An irrigation system with a bar of sprinklers with different flow rates was used. Gas exchange, vegetation indices (measured using a hyperspectral sensor embedded in a drone), yield and grain quality were evaluated. Water stress had different effects on gas exchange, vegetation indices, grain yield and chemical composition among the cultivars. Embrapa cultivar BRS 7280 Roundup ready (RR) and Nidera cultivar NA 5909 RG (glyphosate resistant) are yield stable and have a greater tolerance to drought. BRS 7280RR showed a higher tolerance to drought and higher water use efficiency (WUE) than all other tested cultivars. Vegetation indices, such as the NDVI (Normalized Difference Vegetation Index), correlated with the morphophysiological traits, such as plant height, were the most responsive variables to water stress. The NDVI can be used to predict soybean yield as a tool in a selection program under drought.

Integration of metabolomics and transcriptomics data to further characterize Gliricidia sepium (Jacq.) Kunth under high salinity stress.

Soil salinity is one abiotic stress that threatens agriculture in more than 100 countries. Gliricidia [Gliricidia sepium (Jacq.) Kunth] is a multipurpose tree known for its ability to adapt to a wide range of soils; however, its tolerance limits and responses to salt stress are not yet well understood. In this study, after characterizing the morphophysiological responses of young gliricidia plants to salinity stress, leaf metabolic and transcription profiles were generated and submitted to single and integrated analyses. RNA from leaf samples were subjected to RNA sequencing using an Illumina HiSeq platform and the paired-end strategy. Polar and lipidic fractions from leaf samples were extracted and analyzed on an ultra-high-performance liquid chromatography (UHPLC) coupled with electrospray ionization quadrupole time-of-flight high-resolution mass spectrometry (MS) system. Acquired data were analyzed using the OmicsBox, XCMS Online, MetaboAnalyst, and Omics Fusion platforms. The substrate salinization protocol used allowed the identification of two distinct responses to salt stress: tolerance and adaptation. Single analysis on transcriptome and metabolome data sets led to a group of 5,672 transcripts and 107 metabolites differentially expressed in gliricidia leaves under salt stress. The phenylpropanoid biosynthesis was the most affected pathway, with 15 metabolites and three genes differentially expressed. Results showed that the differentially expressed metabolites and genes from this pathway affect mainly short-term salt stress (STS). The single analysis of the transcriptome identified 12 genes coding for proteins that might play a role in gliricidia response at both STS and long-term salt stress (LTS). Further studies are needed to reveal the mechanisms behind the adaptation response.

Expression analysis of miRNAs and their putative target genes confirm a preponderant role of transcription factors in the early response of oil palm plants to salinity stress.

BACKGROUND: Several mechanisms regulating gene expression contribute to restore and reestablish cellular homeostasis so that plants can adapt and survive in adverse situations. MicroRNAs (miRNAs) play roles important in the transcriptional and post-transcriptional regulation of gene expression, emerging as a regulatory molecule key in the responses to plant stress, such as cold, heat, drought, and salt. This work is a comprehensive and large-scale miRNA analysis performed to characterize the miRNA population present in oil palm (Elaeis guineensis Jacq.) exposed to a high level of salt stress, to identify miRNA-putative target genes in the oil palm genome, and to perform an in silico comparison of the expression profile of the miRNAs and their putative target genes. RESULTS: A group of 79 miRNAs was found in oil palm, been 52 known miRNAs and 27 new ones. The known miRNAs found belonged to 28 families. Those miRNAs led to 229 distinct miRNA-putative target genes identified in the genome of oil palm. miRNAs and putative target genes differentially expressed under salinity stress were then selected for functional annotation analysis. The regulation of transcription, DNA-templated, and the oxidation-reduction process were the biological processes with the highest number of hits to the putative target genes, while protein binding and DNA binding were the molecular functions with the highest number of hits. Finally, the nucleus was the cellular component with the highest number of hits. The functional annotation of the putative target genes differentially expressed under salinity stress showed several ones coding for transcription factors which have already proven able to result in tolerance to salinity stress by overexpression or knockout in other plant species. CONCLUSIONS: Our findings provide new insights into the early response of young oil palm plants to salinity stress and confirm an expected preponderant role of transcription factors - such as NF-YA3, HOX32, and GRF1 - in this response. Besides, it points out potential salt-responsive miRNAs and miRNA-putative target genes that one can utilize to develop oil palm plants tolerant to salinity stress.

Metabolic effect of drought stress on the leaves of young oil palm (Elaeis guineensis) plants using UHPLC-MS and multivariate analysis.

The expansion of the oil palm in marginal areas can face challenges, such as water deficit, leading to an impact on palm oil production. A better understanding of the biological consequences of abiotic stresses on this crop can result from joint metabolic profiling and multivariate analysis. Metabolic profiling of leaves was performed from control and stressed plants (7 and 14 days of stress). Samples were extracted and analyzed on a UHPLC-ESI-Q-TOF-HRMS system. Acquired data were processed using XCMS Online and MetaboAnalyst for multivariate and pathway activity analysis. Metabolism was affected by drought stress through clear segregation between control and stressed groups. More importantly, metabolism changed through time, gradually from 7 to 14 days. The pathways most affected by drought stress were: starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, and glycine, serine and threonine metabolism. The analysis of the metabolic profile were efficient to correlate and differentiate groups of oil palm plants submitted to different levels of drought stress. Putative compounds and their affected pathways can be used in future multiomics analysis.

A procedure for maize genotypes discrimination to drought by chlorophyll fluorescence imaging rapid light curves.

BACKGROUND: Photosynthesis can be roughly separated into biochemical and photochemical processes. Both are affected by drought and can be assessed by non-invasive standard methods. Gas exchange, which mainly assesses the first process, has well-defined protocols. It is considered a standard method for evaluation of plant responses to drought. Under such stress, assessment of photochemical apparatus by chlorophyll fluorescence needs improvement to become faster and reproducible, especially in growing plants under field conditions. For this, we developed a protocol based on chlorophyll fluorescence imaging, using a rapid light curve approach. RESULTS: Almost all parameters obtained by rapid light curves have shown statistical differences between control and drought stressed maize plants. However, most of them were affected by induction processes, relaxation rate, and/or differences in chlorophyll content; while they all were influenced by actinic light intensity on each light step of light curve. Only the normalized parameters related to photochemical and non-photochemical quenching were strongly correlated with data obtained by gas exchange, but only from the light step in which the linear electron flow reached saturation. CONCLUSIONS: The procedure developed in this study for discrimination of plant responses to water deficit stress proved to be as fast, efficient and reliable as the standard technique of gas exchange in order to discriminate the responses of maize genotypes to drought. However, unlike that, there is no need to perform daily and time consuming calibration routines. Moreover, plant acclimation to the dark is not required. The protocol can be applied to plants growing in both controlled conditions and full sunlight in the field. In addition, it generates parameters in a fast and accurate measurement process, which enables evaluating several plants in a short period of time.

Improved Genetic Transformation of Sugarcane (Saccharum spp.) Embryogenic Callus Mediated by Agrobacterium tumefaciens.

Sugarcane (Saccharum spp.) is a monocotyledonous semi-perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several limitations due to its complex polyploid and aneuploid genome. However, improvement by biotechnological engineering is currently the most promising alternative to introduce economically important traits. In this work, we present an improved protocol for Agrobacterium tumefaciens-mediated transformation of commercial sugarcane hybrids using immature top stalk-derived embryogenic callus cultures. The callus cultures are transformed with preconditioned A. tumefaciens carrying a binary vector that encodes expression cassettes for a gene of interest and the bialaphos resistance gene (bar confers resistance to glufosinate-ammonium herbicide). This protocol has been used to successfully transform a commercial sugarcane cultivar, SP80-3280, highlighting: (i) reduced recalcitrance and oxidation; (ii) high yield of embryogenic callus; (iii) improved selection; and (iv) shoot regeneration and rooting of the transformed plants. Altogether, these improvements generated a transformation efficiency of 2.2%. This protocol provides a reliable tool for a routine procedure for sugarcane improvement by genetic engineering. © 2017 by John Wiley & Sons, Inc.

Induced over-expression of AtDREB2A CA improves drought tolerance in sugarcane.

Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and, in some cases, yield losses caused by drought are nearly 50%. DREB proteins play vital regulatory roles in abiotic stress responses in plants. The transcription factor DREB2A interacts with a cis-acting DRE sequence to activate the expression of downstream genes that are involved in drought-, salt- and heat-stress response in Arabidopsis thaliana. In the present study, we evaluated the effects of stress-inducible over-expression of AtDREB2A CA on gene expression, leaf water potential (ΨL), relative water content (RWC), sucrose content and gas exchanges of sugarcane plants submitted to a four-days water deficit treatment in a rhizotron-grown root system. The plants were also phenotyped by scanning the roots and measuring morphological parameters of the shoot. The stress-inducible expression of AtDREB2A CA in transgenic sugarcane led to the up-regulation of genes involved in plant response to drought stress. The transgenic plants maintained higher RWC and ΨL over 4 days after withholding water and had higher photosynthetic rates until the 3rd day of water-deficit. Induced expression of AtDREB2A CA in sugarcane increased sucrose levels and improved bud sprouting of the transgenic plants. Our results indicate that induced expression of AtDREB2A CA in sugarcane enhanced its drought tolerance without biomass penalty.