Gene Editing for Plant Resistance to Abiotic Factors: A Systematic Review.

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.

Multiplex CRISPR/Cas9-mediated knockout of the phytoene desaturase gene in Coffea canephora.

Coffea canephora (2n = 2x = 22 chromosomes) is a species with extensive genetic diversity and desirable agronomic traits for coffee breeding programs. However, obtaining a new coffee cultivar through conventional breeding techniques may require more than 30 years of crossing cycles and selection, which hampers the effort of keeping up with market demands and rapidly proposing more resilient to climate change varieties. Although, the application of modern biotechnology tools such as precision genetic engineering technologies may enable a faster cultivar development process. Therefore, we aimed to validate the CRISPR/Cas9 system to generate mutations on a selected genotype of C. canephora, the clone 14. Embryogenic calli and a multiplex binary vector containing two sgRNAs targeting different exons of the CcPDS gene were used. The sgRNAs were under the C. canephora U6 promoter regulation. The target gene encodes phytoene desaturase, an enzyme essential for photosynthesis involved in ß-carotene biosynthesis. Somatic seedlings and embryos with albino, variegated and green phenotypes regenerated after Agrobacterium tumefaciens-mediated genetic transformation were analyzed by verifying the insertion of the Cas9 gene and later by sequencing the sgRNAs target regions in the genome of Robusta modified seedlings. Among them, 77% had the expected mutations, and of which, 50% of them had at least one target with a homozygous mutation. The genotype, temperature of co-cultivation with the bacteria, and light intensity used for subsequent embryo regeneration appeared to strongly influence the successful regeneration of plants with a mutated CcPDS gene in the Coffea genus.

The SAUR gene family in coffee: genome-wide identification and gene expression analysis during somatic embryogenesis.

BACKGROUND: Small auxin-up RNA (SAUR) genes form a wide family supposedly involved in different physiological and developmental processes in plants such as leaf senescence, auxin signaling and transport, hypocotyl development and tolerance to abiotic stresses. The transcription of SAUR genes is quickly induced by auxins, a group of phytohormones of major importance on embryo development. To better understand the distribution and expression profile of such still not explored family in Coffea sp., especially during the development of somatic embryogenesis (SE), SAUR members were characterized in silico using the available Coffea canephora genome data and analyzed for gene expression by RT-qPCR in C. arabica embryogenic samples. METHODS AND RESULTS: Over C. canephora genome 31 CcSAURs were distributed by 11 chromosomes. Out of these 31 gene members, 5 SAURs were selected for gene expression analysis in C. arabica embryogenic materials. CaSAUR12 and CaSAUR18 were the members highly expressed through almost all plant materials. The other genes had more expression in at least one of the developing embryo stages or plantlets. The CaSAUR12 was the only member to exhibit an increased expression in both non-embryogenic calli and the developing embryo stages. CONCLUSION: The identification of SAUR family on C. canephora genome followed by the analysis of gene expression profile across coffee somatic embryogenesis process on C. arabica represents a further additional step towards a better comprehension of molecular components acting on SE. Along with new research about this gene family such knowledge may support studies about clonal propagation methods via somatic embryogenesis to help the scientific community towards improvements into coffee crop.

A new species of Oecleus Stål (Hemiptera: Fulgoroidea: Cixiidae) from coconut in Brazil.

A new species of cixiid planthopper (Hemiptera: Fulgoroidea) in the genus Oecleus Stål, Oecleus sergipensis n. sp., is described from Sergipe State, Brazil. This new taxon is associated with coconut (Cocos nucifera L.) and date palm (Phoenix L). The species was detected in Auchenorrhyncha surveys to find potential vectors of lethal yellowing type syndrome. This is the first report of the genus Oecleus in Brazil. Sequence data from the mitochondrial cytochrome c oxidase subunit I (COI) barcoding region was obtained and accessioned into GenBank.

Genetic polymorphism among 14 elite Coffea arabica L. cultivars using RAPD markers associated with restriction digestion

Knowledge of the genetic variability among genotypes is important for the transfer of useful genes and to maximize the use of available germplasm resources. This study was carried out to assess the genetic variability of 14 elite Coffea arabica cultivars using random amplified polymorphic DNA (RAPD) associated with a prior digestion of genomic DNA with restriction endonucleases. The accessions were obtained from the Coffea collection maintained at the Instituto Agronomico do Parana (IAPAR), located in Londrina, Parana, Brazil. Twenty-four informative RAPD primers, used in association with restriction enzymes, yielded 330 reproducible and scorable DNA bands, of which 224 (68 per cent) were polymorphic. The amplified products were used to estimate the genetic variability using Dice's similarity coefficient. The data matrix was converted to a dendrogram and a three-dimensional plot using principal coordinate analysis. The accessions studied were separated into clusters in a manner that was consistent with the known pedigree. The associations obtained in the dendrogram and in the principal coordinate analysis plot suggest the probable origin of the Kattimor cultivar. The RAPD technique associated with restriction digestion was proved to be a useful tool for genetic characterization of C. arabica genotypes making an important contribution to the application of molecular markers to coffee breeding