As a member of Convolvulaceae family, sweetpotato (Ipomoea batatas L.) is an important crop for food security. As one of the top three vegetable crops grown in Mississippi, one major limitation to sweetpotato production is the cumulative effect of virus infection leading to cultivar decline and yield losses. To produce virus-tested sweetpotato seedlings, we developed meristem-tip culture technology combined with heat treatment to provide farmers with healthy propagating materials that are free of detectable viruses. In this study, totally 30 lines of sweetpotato have been collected in Mississippi and beyond. The plants were first examined with the infection of five of the most prevalent viruses by using nucleic acid-based polymerase chain reaction (PCR) and reverse-transcription PCR (RT-PCR) techniques, which showed high sensitivity and confirmation at the genomic level of viral species and strains. Primers targeting to conserved regions of the know sweetpotato viruses were used for this nucleic acid based detection. The effective protocols for sweetpotato viral detection and viral removal were well developed in this study. The optimized protocols have been used for the purpose of viral detection and eradicating from elite sweetpotato lines in Mississippi. Virus-free planting material has been propagated in Agriculture Research Station of Alcorn State University for performance evaluation.
Bigleaf hydrangea, Hydrangea macrophylla, is a popular ornamental shrub beloved worldwide for its large colorful inflorescences. As of 2019, bigleaf hydrangea topped $155 million in total sales in the United States. However, diseases impact the health, appearance and ultimately the salability of bigleaf hydrangea. One such disease is powdery mildew, caused by the fungus Golovinomyces orontii. Powdery mildew is a biotrophic obligate, which means that it will only grow on a living host, making it particularly hard to investigate. Inoculating plants via tapping infected leaves against clean leaves is the most common inoculation method; however, this method results in an unknown amount of inoculum and inconsistent infection. Quantifiable and consistent methods are needed to accurately and reproducibly study powdery mildew disease of bigleaf hydrangea. The purpose of this experiment was to compare two inoculation methods, an inoculation box and spray inoculation method, for inducing powdery mildew disease in bigleaf hydrangea. For the box inoculation, 6 infected leaves per plant were tapped above a 48-micron mesh and gently dusted through to land on the plant surface. A still air chamber was designed to be set over the box so that powdery mildew does not get disturbed by air currents. For the spray inoculation, 6 infected leaves per plant were combined into a spray by rinsing the leaves with deionized water, adding Tween 20 to aid with even dispersion and applied to the plant using a spray bottle. Powdery mildew was then evaluated on a scale of 0-100% and the area under disease progress curve (AUDPC) was calculated. The final disease severity of hydrangea inoculated via the box and spray method after 4 weeks of observation were 10.0 and 20.0%, respectively. These results will provide valuable information on more consistent inoculation methods using powdery mildew.
Blueberry (Vaccinium spp.) is one of the most economically important woody plant species because its fruit is rich in antioxidants such as anthocyanin, which offer beneficial effects on human health. Thus, the demand for blueberry production and for the development of novel elite cultivars has been continuously increasing. Although new blueberry cultivars have been developed by cross breeding for more than a century, the conventional breeding approaches are time-consuming and labor-intensive due to the associated characteristics that hinder efficient breeding such as long juvenile phase, polyploidy, and heterozygosity. Genetic engineering offers a promising approach to confer a desirable trait to elite cultivars. In particular, the recently developed genome editing technology enables precise modifications of plant genomes. We aim to apply genome editing to improve agronomically important traits in blueberry, specifically anthocyanin and sugar content. Towards the development of high anthocyanin blueberry, we targeted an anthocyanin repressor gene, VcMYBC2. So far, we successfully obtained 3 lines with all-alleles of VcMYBC2 were mutated. An increase in anthocyanin content in fruit is expected in the mybc2 mutants. Regarding sugar content, we targeted invertase inhibitor (INVINH) gene that is involved in sugar metabolism. Invertase promotes sucrose unloading in the fruit by maintaining a gradient of sucrose concentration between source leaves and fruits, while INVINH represses this process. Thus, higher sugar accumulation is expected in invinh mutants. Three transgenic lines harboring CRISPR-Cas9 vectors targeting VcINVINH genes that are highly expressed in fruit tissue, were obtained. The mutated allele frequencies of the mutants ranged from 21-67%. Additionally, we generated transgenic blueberries overexpressing FLOWERING LOCUS T (FT), a mobile florigen signal gene that induces flowering. A previous study demonstrated that blueberry scions grafted to FT-overexpressing lines could show early flowering. We thus assume that the mybc2 and invinh mutants may flower earlier when they are grafted onto the FT overexpressing rootstock, which will accelerate our fruit phenotype evaluations. The FT-overexpressing rootstock may also be utilized to facilitate the production of null-segregant mutants.
Sulfur (S) is an essential nutrient present in nearly all proteins as well as numerous secondary metabolites and critical biochemical intermediates. Research across many crop species has demonstrated that post-stress S fertilization can improve plant recovery from various abiotic and biotic stresses. Furthermore, reported findings from these studies suggest that, in the absence of stress, enhanced S fertilization improves the expression of enzymes and metabolites with known stress-defense functions. As part of a new project seeking to test the impact of pre-stress tomato S status on heat stress resilience, we conducted a genome-wide mining and characterization study of the tomato sulfate transporter (SULTR) gene family. Following the typical steps in similar genome mining studies, we identified 14 putative SlSULTR genes through a BLASTp search of known A. thaliana SULTR proteins in the Phytozome database. Then, phylogenetic relationships between the putative SlSULTR genes were characterized using a neighbor-joining tree method after aligning the sequences with the MUSCLE algorithm in MEGA v12. The putative genes were also characterized for their exon-intron structure and intron cycle, which were retrieved from Phytozome and the GSDS server, respectively. Characterization of the putative SlSULTR proteins included transmembrane topology prediction using the SCAMPI2 server and motif analysis using MEME Suite v5.5.7. De novo motif discovery using the MEME algorithm produced 10 amino acid motifs with E-values under 1E-164. These de novo motifs were scanned against Ensembl’s A. thaliana protein database using the MAST algorithm which returned 27 A. thaliana transcripts, each from an AtSULTR gene and with highly significant motif similarities. To characterize the regulation of the putative SlSULTR genes, we searched for SlSULTR-targeted miRNAs in the psRNATarget database as well as identified cis-regulatory motifs in the 1000 bp upstream region of each genomic sequence through the PlantCARE server. Predicted miRNA were from various miRNA families known for their responses to biotic and abiotic stresses and their regulation of nutrient uptake and distribution, plant development, disease resistance, and signal transduction. Lastly, the identified cis-regulatory motifs were largely associated with plant light response but also included motifs associated with general stress and hormone signal response. This work represents the first genomic mining study of tomato SULTR genes. Our phylogenetic and amino acid motif results provide strong evidence that the selected sequences operate as SULTR genes in tomatoes, and the identified miRNA targets and cis-regulatory sequences reflect established research demonstrating the role of S in plant stress response.
Agrobacterium-mediated transformation is widely used in plant genetic engineering. This method involves both Agrobacterium infection and plant regeneration. In this context, establishing an efficient plant regeneration system is a critical prerequisite for genetic engineering in plants. This study aims to identify highbush blueberry (Vaccinium corymbosum L.) cultivars suitable for genetic transformation. Furthermore, we seek to elucidate the molecular and genetic factors that determine genotype-dependent shoot regeneration capacity by utilizing the diverse genetic background in highbush blueberries. Additionally, this study explores cultivar-specific differences in Agrobacterium susceptibility, which remain unexplored in highbush blueberries. Regeneration from leaf explants of 15 highbush blueberry cultivars was investigated on media containing 1.0 mg/L TDZ or 1.0 mg/L TDZ and 0.5 mg/L NAA. There was considerable variation in callus formation and regeneration rates, the number of regenerated shoots, the time required for regeneration, indicating that regeneration in highbush blueberry is highly genotype-dependent. The regeneration rate was high (>75%) in ‘Blue Muffin’, ‘Legacy’, ‘Gulfcoast’ and ‘Georgiagem’. However, ‘Georgiagem’ required three additional weeks for shoot regeneration from the time of meristem formation compared to the other three high regeneration cultivars. All four northern highbush cultivars exhibited low regeneration rates (
Protein phosphatases, particularly PP2C families, are vital regulators of cellular activity through the removal of phosphate groups from proteins. Numerous biological processes, such as hormone signaling, reactions to heat and drought stress, and abiotic stress tolerance, are impacted by this dephosphorylation process. This study presents comprehensive genomic analysis, evolutionary assessment and transcript profiling of the PP2C gene family in sweet potato, a crop of major agricultural and nutritional importance. A total of 74 PP2C genes have been identified in sweet potato. Among them, 7 gene pairs were identified as segmental duplication while 5 pairs as tandem duplications. Phylogenetic analysis grouped them into distinct clusters, indicating potential functional divergence. Gene structure analysis provided insights into the arrangement of coding and non-coding regions. Motif and domain analysis highlighted conserved protein sequences aiding functional predictions. Synteny analysis compared genomic regions across species identifying homologous genes and tracing genome evolution. The study of cis-regulatory elements in promoters helped map gene regulations. Gene expression analysis is currently being conducted to determine the transcript levels of PP2C during salinity and drought stress. The results provide a basis for additional validation of the roles of the PP2C gene in sweet potatoes and advance our knowledge of the evolutionary background and functional significance of PP2C in biotic and abiotic stress response.
