Cotton plants overexpressing the Bacillus thuringiensis Cry23Aa and Cry37Aa binary-like toxins exhibit high resistance to the cotton boll weevil (Anthonomus grandis).

The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of ß-pore-forming toxins (ß-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin's mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.

MicroRNAs expression profiles in early responses to different levels of water deficit in Setaria viridis.

Water deficit is a major constraint for crops of economic importance in almost all agricultural regions. However, plants have an active defense system to adapt to these adverse conditions, acting in the reprogramming of gene expression responsible for encoding microRNAs (miRNAs). These miRNAs promote the regulation to the target gene expression by the post-transcriptional (PTGS) and transcriptional gene silencing (TGS), modulating several pathways including defense response to water deficit. The broader knowledge of the miRNA expression profile and its regulatory networks in response to water deficit can provide evidence for the development of new biotechnological tools for genetic improvement of several important crops. In this study, we used Setaria viridis accession A10.1 as a C4 model plant to widely investigate the miRNA expression profile in early responses to different levels of water deficit. Ecophysiological studies in Setaria viridis under water deficit and after rewatering demonstrated a drought tolerant accession, capable of a rapid recovery from the stress. Deep small RNA sequencing and degradome studies were performed in plants submitted to drought to identify differentially expressed miRNA genes and their predicted targets, using in silico analysis. Our findings showed that several miRNAs were differentially modulated in response to distinctive levels of water deficit and after rewatering. The predicted mRNA targets mainly corresponded to genes related to cell wall remodeling, antioxidant system and drought-related transcription factors, indicating that these genes are rapidly regulated in early responses to drought stress. The implications of these modulations are extensively discussed, and higher-effect miRNAs are suggested as major players for potential use in genetic engineering to improve drought tolerance in economically important crops, such as sugarcane, maize, and sorghum. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01226-z.

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.

Characterization of sugarcane (Saccharum spp.) leaf senescence: implications for biofuel production.

BACKGROUND: Second-generation ethanol (2G-bioethanol) uses lignocellulosic feedstocks for ethanol production. Sugarcane is one among the most suitable crops for biofuel production. Its juice is extracted for sugar production, while sugarcane bagasse, straw, and senescing leaves are considered industrial waste. Senescence is the age-dependent deterioration of plant cells, ultimately leading to cell death and completion of the plant life cycle. Because senescing leaves may also be used for biofuel production, understanding the process of natural senescence, including remobilization of nutrients and its effect on cell walls can provide useful information for 2G-bioethanol production from sugarcane leaves. RESULTS: The natural senescence process in leaves of the commercial sugarcane cultivar RB867515 was investigated. Senescence was characterized by strong reduction in photosynthetic pigments content, remobilization of the nutrients N, P, K, B, Cu, Fe, and Zn, and accumulation of Ca, S, Mg, B, Mn, and Al. No significant changes in the cell-wall composition occurred, and only small changes in the expression of cell wall-related genes were observed, suggesting that cell walls are preserved during senescence. Senescence-marker genes, such as SAG12-like and XET-like genes, were also identified in sugarcane and found to be highly expressed. CONCLUSIONS: Our study on nutrient remobilization under senescence in a vigorous sugarcane cultivar can contribute to the understanding on how nutrient balance in a high-yielding crop is achieved. In general, neutral monosaccharide profile did not change significantly with leaf senescence, suggesting that senescing leaves of sugarcane can be as a feedstock for biofuel production using pretreatments established for non-senescing leaves without additional efforts. Based on our findings, the potential biotechnological applications for the improvement of sugarcane cultivars are discussed.

Influência da temperatura de armazenamento na qualidade das inflorescências de estrelítzia (Strelitzia reginae Banks ex Aiton) / Influence of storage temperature on quality of inflorescences strelitzia (Strelitzia reginae Banks ex Aiton)

O presente trabalho tem por objetivo de estudar a influência da temperatura de armazenamento (câmara fria e temperatura ambiente) na qualidade das inflorescências de estrelítzia. Escapos florais foram selecionados, etiquetados e descartados quanto à presença de danos mecânicos, doenças e/ou pragas. Transcorrido esse período, foram transferidos para recipientes, onde foram submetidos a dois experimentos. No experimento 1, escapos foram colocados em baldes contendo água da rede pública e levadas para câmara fria a 7,5 ºC e UR de 90%, por um período de doze dias. No experimento 2, os escapos foram mantidos nas mesmas condições, porém em temperatura ambiente por um período de seis dias. Nos dois experimentos, as análises visuais: coloração, brilho, manchas (através da atribuição de notas), abertura e queda de floretes (contagem) foram avaliadas em intervalo de quatro dias em câmara fria e a cada 48 horas em condições de temperatura ambiente. A sépala foi o órgão que apresentou maior perda na coloração. A variável brilho apresentou o mesmo comportamento em relação aos dois experimentos. Maiores incidências de manchas nas inflorescências ocorreram em temperatura ambiente. Os escapos apresentaram aumento no número de floretes abertos em câmara fria. Essa tendência não ocorreu em temperatura ambiente. Não foram observados diferenças na queda de floretes. Conclui-se que a temperatura de armazenamento não contribui para a qualidade pós-colheita de estrelítzia.

This work was based to study the influence of the storage temperature (cold and room temperature) in the quality of inflorescences strelitzia. The scapes were selected, labeled and there were zero problems concerning mechanical damage, disease and/or plagues. Subsequently this period, the scapes were moved randomly to recipients with water, in which two postharvest trials were conducted. In experiment 1, the flower scapes were placed in buckets with water from public supply and sanitation department and taken to a cold room at temperature of 7.5 ºC and RH of 90%, for a twelve day period. For the experiment 2, were kept under the same conditions but at room temperature for a period of six days. In both experiments, the visual analysis: color, gloss, stains (by assigning notes), opening and drop florets (count) were evaluated at intervals of four days in cold and every 48 hours at ambient temperature conditions. In both experiments, the visual analysis: color, gloss, stains (by assigning notes), opening and drop florets (count) were evaluated at intervals of four days in cold and every 48 hours at ambient temperature conditions. The sepal is the organ that showed greater loss in coloration. The variable gloss showed the same pattern for the two experiments. Incidences of stains on the inflorescences occurred in patches at room temperature. The scapes increased number of florets open in cold. This tendency did not occur at room temperature. No were observed differences in the fall of florets. Conclude that the storage temperature does not contribute to postharvest quality of strelitzia.