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Agricultural crops are exposed to various abiotic stresses, such as salinity, water deficits, temperature extremes, floods, radiation, and metal toxicity. To overcome these challenges, breeding programs seek to improve methods and techniques. Gene editing by Clustered Regularly Interspaced Short Palindromic Repeats—CRISPR/Cas—is a versatile tool for editing in all layers of the central dogma with focus on the development of cultivars of plants resistant or tolerant to multiple biotic or abiotic stresses. This systematic review (SR) brings new contributions to the study of the use of CRISPR/Cas in gene editing for tolerance to abiotic stress in plants. Articles deposited in different electronic databases, using a search string and predefined inclusion and exclusion criteria, were evaluated. This SR demonstrates that the CRISPR/Cas system has been applied to several plant species to promote tolerance to the main abiotic stresses. Among the most studied crops are rice and Arabidopsis thaliana, an important staple food for the population, and a model plant in genetics/biotechnology, respectively, and more recently tomato, whose number of studies has increased since 2021. Most studies were conducted in Asia, specifically in China. The Cas9 enzyme is used in most articles, and only Cas12a is used as an additional gene editing tool in plants. Ribonucleoproteins (RNPs) have emerged as a DNA-free strategy for genome editing without exogenous DNA. This SR also identifies several genes edited by CRISPR/Cas, and it also shows that plant responses to stress factors are mediated by many complex-signaling pathways. In addition, the quality of the articles included in this SR was validated by a risk of bias analysis. The information gathered in this SR helps to understand the current state of CRISPR/Cas in the editing of genes and noncoding sequences, which plays a key role in the regulation of various biological processes and the tolerance to multiple abiotic stresses, with potential for use in plant genetic improvement programs.

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is the most limiting factor in the banana agribusiness worldwide. Therefore, studies regarding pathogen attack mechanisms, and especially host defense responses, in this pathosystem are of utmost importance for genetic breeding programs in the development of Foc-resistant banana cultivars. In this study, analysis at the molecular, histological and histochemical levels of the Musa spp. x Foc interaction was performed. Three Foc isolates representative of race 1 (R1), subtropical race 4 (ST4) and isolate 229A, which is a putative ST4, were inoculated in two Prata-type cultivars (Prata-Anã and BRS Platina) and one cultivar of the Cavendish type (Grand Naine). Of seven genes related to plant–pathogen interactions, five were overexpressed in ‘BRS Platina’ 12 h after inoculation (HAI) with Foc R1 and ST4 but had reduced or negative expression after inoculation with Foc 229A, according to RT–qPCR analyses. While hyphae, mycelia and spores of the Foc 229A isolate grow towards the central cylinder of the Grand Naine and Prata-Anã cultivars, culminating in the occlusion of the xylem vessels, the BRS Platina cultivar responds with increased presence of cellulose, phenolic compounds and calcium oxalate crystals, reducing colonization within 30 days after inoculation (DAI). In general, these data indicate that the cultivar BRS Platina has potential for use in banana-breeding programs focused on resistance to Foc tropical race 4 (TR4) and in aggregating information on the virulence relationships of the Foc pathogen and the defense responses of banana plants after infection.

Phytoene desaturase (PDS) is a plant enzyme involved in carotenoid biosynthesis. The PDS gene has been used as a selective marker for genome editing in several plant species, including banana (Musa spp.). Its knockout promotes dwarfism and albinism, characteristics that are easily recognizable and highly favorable. In Musa spp., the A genome increases fruit production and quality, whereas the B genome is associated with tolerance to biotic and abiotic stresses. The objective of this study was to identify a molecular marker in the PDS gene to easily discriminate the A and B genomes of banana. A 2166 bp fragment for the “PDSMa” marker was identified as polymorphic for the A genome (identification accuracy of 99.33%), whereas ~332 and ~225 bp fragments were detected for the “PDSMb” marker with 100% accuracy using MedCalc software. In this study, we used genotypes with A and B genomes that are used in the genetic improvement of bananas and an accession with the BT genome. It was not possible to differentiate the accession with the BT genome from the others, suggesting that the markers do not have the capacity to separate the T genome from the A and B genomes. To the best of our knowledge, this is the first study to use the PDS gene to determine doses of the A genome and identify the B genome in Musa spp., which will aid in evaluating the genomic constitution of banana hybrids and accessions at the seedling stage and accelerating their classification in crop genetic improvement programs.

Bananas are the main fruits responsible for feeding more than 500 million people in tropical and subtropical countries. Black Sigatoka, caused by the fungus Pseudocercospora fijiensis, is one of the most destructive disease for the crop. This fungus is mainly controlled with the use of fungicides; however, in addition to being harmful to human health, they are associated with a high cost. The development of resistant cultivars through crosses of susceptible commercial cultivars is one of the main focuses of banana breeding programs worldwide. Thus, the objective of the present study was to investigate the interaction between Musa sp. and P. fijiensis through the relative expression of candidate genes involved in the defence response to black Sigatoka in four contrasting genotypes (resistant: Calcutta 4 and Krasan Saichon; susceptible: Grand Naine and Akondro Mainty) using quantitative real-time PCR (RT–qPCR) in addition to histological and histochemical analyses to verify the defence mechanisms activated during the interaction. Differentially expressed genes (DEGs) related to the jasmonic acid and ethylene signalling pathway, GDSL-like lipases and pathogenesis-related proteins (PR-4), were identified. The number and distance between stomata were directly related to the resistance/susceptibility of each genotype. Histochemical tests showed the production of phenolic compounds and callosis as defence mechanisms activated by the resistant genotypes during the interaction process. Scanning electron microscopy (SEM) showed pathogenic structures on the leaf surface in addition to calcium oxalate crystals. The resistant genotype Krasan Saichon stood out in the analyses and has potential for use in breeding programs for resistance to black Sigatoka in banana and plantains.

Banana ( .) is a globally significant crop and a staple food in the diet of millions of people. However, commercial cultivars are highly susceptible to Fusarium wilt, a devastating disease caused by f. sp. . Tropical race 4 (TR4) and Subtropical race 4 (STR4) pose significant threats to banana production, including 'Cavendish' (AAA group), with STR4 pathogenic only in subtropical regions. Genetic resistance is the most effective strategy to combat Foc, underscoring the importance of advancing understanding of resistance mechanisms. Here, we identified and validated genes involved in the resistance response to Foc STR4 through RNA-seq and RT-qPCR analyses. Two genotypes were evaluated: 'Calcutta 4' (a resistant wild genotype, AA) and 'Prata-Anã' (a susceptible commercial genotype, AAB). Seedlings of 'Calcutta 4' and 'Prata-Anã' were inoculated with Foc STR4 isolate 218A, and root samples from 'Calcutta 4' were collected at 1, 2, and 4 days after inoculation (DAI) for RNA-seq analysis. Comparative histological studies between the genotypes revealed defence responses, such as callose deposition and phenolic compound production, occurring exclusively in 'Calcutta 4' at 1 and 2 DAI, while colonization by STR4 was observed only in 'Prata-Anã' at 8 and 15 DAI. RNA-seq analysis identified 1416 differentially expressed genes (DEGs) in 'Calcutta 4', based on comparisons between inoculated and non-inoculated control plants, log2FC >2 and <-2, and adjusted p-value for FDR at <0.05, with a rapid upregulation of 752 DEGs at 2 DAI, including genes associated with pattern recognition receptors, chitinases, phytohormones, resistance genes (from the NLR family), TFs, and systemic acquired resistance. Functional pathway analysis highlighted coordinated defence responses in 'Calcutta 4' to STR4. Together with functional validation of selected genes via RT-qPCR, these findings provide a foundation for the application of candidate genes in genetic improvement via introgression or gene-editing approaches. Given the close phylogenetic relationship between Foc STR4 and TR4, introgression of defense-related genes also holds promise for developing varieties that are resistant to both race 4 pathogens, relevant for mitigating the global impact of Fusarium wilt epidemics on banana production.

Bananas, a staple food globally and a key agricultural commodity, face a severe threat from the fungus Fusarium oxysporum f. sp. cubense (Foc), significantly impacting production. Genetic improvement to develop resistant cultivars stands out as a crucial strategy to mitigate this disease. This study focused on assessing and recommending enhanced diploid banana varieties for resistance against Foc subtropical race 4 (ST4) and Foc race 1 (R1). Twenty-four improved diploids developed by Embrapa, Brazil, underwent evaluation. Utilizing a scale for internal symptoms at 90 days after inoculation, genotypes were categorized from highly resistant to highly susceptible based on the internal symptom index. The diploid M53 exhibited high resistance to Foc R1 and resistance to Foc ST4, while only the diploid CNPMF 0534 demonstrated complete resistance to both R1 and ST4, with resistance to the latter likely associated with penetration, primarily due to the presence of callose. These findings provide valuable insights for banana and plantain breeding programs, offering selected diploids for crossbreeding with commercial cultivars to develop new, resistant genotypes against Foc.

Fusarium wilt is a soil borne fungal disease that has devastated banana production in plantations around the world. Most Cavendish-type bananas are susceptible to strains of Fusarium oxysporum f. sp. cubense (Foc) belonging to the Subtropical Race 4 (STR4) and Tropical Race 4 (TR4). The wild banana diploid Musa acuminata ssp. malaccensis (AA, 2n = 22) carries resistance to Foc TR4. A previous study using segregating populations derived from M. acuminata ssp. malaccensis identified a quantitative trait locus (QTL) (12.9 cM) on the distal part of the long arm of chromosome 3, conferring resistance to both Foc TR4 and STR4. An SNP marker, based on the gene Macma4_03_g32560 of the reference genome ‘DH-Pahang’ v4, detected the segregation of resistance to Foc STR4 and TR4 at this locus. Using this marker, we assessed putative TR4 resistance sources in 123 accessions from the breeding program in Brazil, which houses one of the largest germplasm collections of Musa spp. in the world. The resistance marker allele was detected in a number of accessions, including improved diploids and commercial cultivars. Sequencing further confirmed the identity of the SNP at this locus. Results from the marker screening will assist in developing strategies for pre-breeding Foc TR4-resistant bananas. This study represents the first-ever report of marker-assisted screening in a comprehensive collection of banana accessions in South America. Accessions carrying the resistance marker allele will be validated in the field to confirm Foc TR4 resistance.

The objective of this systematic review (SR) was to select studies on the activity of glycerol as a molecule that induces disease resistance in plants. We sought to evaluate articles deposited in five electronic databases using a search string and predefined inclusion and exclusion criteria. The most studied crops are Arabidopsis thaliana, Glycine max, and Coffea spp. The most commonly cited biotic agents include Pseudomonas syringae, Blumeria graminis, and Colletotrichum higginsianum. Numerous doses of glycerol were studied, and concentrations ranged from 0.004 to 9.21%, with a 3% concentration of glycerol being considered most effective for most plant species, where greater resistance was observed with increased glycerol-3-phosphate (G3P) and decreased oleic acid levels. The main means of application of the product were spraying and immersion. The SR also revealed the evaluation of resistance-inducing genes, such as PR proteins (PR-1, PR2, PR-5, etc.), HPS70, HSP90, SCAM4, and Tapr1, among others. The information collected in this SR helps to understand the state of the art on the use of glycerol as a molecule inducing resistance against biotic stressors to understand the mechanisms involved in most host–pathogen relationships. This information will be useful in plant breeding programs and for growers/producers.

Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases affecting banana cultivation worldwide. Although Foc tropical race 4 (TR4) has not yet been identified in Brazilian production areas, the damage caused by races 1 and subtropical 4 is the main cause of production losses, especially affecting cultivars of the Prata subgroup. Thus, the induction of somaclonal variation is a promising strategy in biotechnology to generate genetic variability and develop resistant varieties. This study aimed to induce somaclonal variation in the Prata Catarina cultivar (AAB genome) using successive subcultures in Murashige and Skoog (MS) medium enriched with the plant regulator Thiadizuron (TDZ) at two concentrations: 1 and 2 mg/L. After evaluating the symptoms, we selected 13 resistant somaclones that were not infected by the fungus. Histochemical and histological analyses of the somaclones indicated possible defense mechanisms that prevented colonization and/or infection by Foc, such as intense production of phenolic compounds and the presence of cellulose and callose in the roots. Some somaclones showed no pathogen structures in the xylem-conducting vessels, indicating possible pre-penetration resistance. Furthermore, molecular studies indicated that the genetic alterations in the somaclones may have induced resistance to Foc without compromising the agronomic characteristics of the commercial genotype.

Yellow Sigatoka, caused by the fungus Pseudocercospora musae, represents one of the most challenging diseases in bananas, which is aggravated due to the genetic variability of this pathogen. The main objective of this study was to characterize the infection process of P. musae in two banana cultivars with different levels of resistance and to quantify the expression of resistance genes. Inoculated and non-inoculated leaf samples of the two cultivars, Yangambi km 5—resistant and Grande Naine—susceptible, were investigated by light microscopy, histochemistry, scanning electron microscopy, and RT-qPCR. The cultivar Grande Naine showed early signs of P. musae infection, including the production of lipophilic substances and phenolic compounds, while the cultivar Yangambi km 5 was not affected. In the Grande Naine cultivar, the protein kinase gene was upregulated, while in the Yangambi km 5, it was repressed. The WAK2 gene was initially upregulated in both cultivars; however, in Grande Naine, it was subsequently repressed, while in Yangambi km 5, it remained downregulated. These findings have the potential to contribute to more effective management strategies in the control of yellow Sigatoka and the development of banana varieties resistant to yellow Sigatoka.