Climate change poses a significant threat to perennial crops like grapevine in the USA, with projections indicating that 50-81% of acreage may become unproductive by 2040. To address this, breeding resilient varieties is imperative. However, traditional breeding for perennials is time-consuming (25-30 years) due to extended evaluation periods. Genome Editing, specifically CRISPR/Cas9, offers targeted modification potential but faces hurdles in perennial crops such as grapevine. These include somaclonal variation, inefficient transformation, transgene removal needs, and industry hesitance. We propose an efficient Genome Editing method for grapevine leveraging the systemic mobility of methylated dicistronic mRNA:tRNA molecules. This approach aims to: 1. Enable genome editing without transgene integration, negating the need for backcrossing and preventing unintended genetic changes. 2. Circumvent in-vitro culture, reducing somaclonal variation risk and preserving existing chimerism. Our method addresses key challenges in Genome Editing adoption for grapevine and could accelerate the development of climate-resilient varieties, crucial for sustainable agriculture in a changing climate.
Tall fescue (Lolium arundinacea Schreb.) and perennial ryegrass (Lolium perenne) are common cool-season turfgrass species. They are widely utilized in home lawns, athletic fields, golf courses, and roadsides. However, these grasses require frequent mowing to maintain lawn quality. Developing low-mowing frequency varieties is highly desirable because it can significantly reduce maintenance costs and fuel consumption. Gibberellin 20-oxidases (GA20ox) are key genes in the gibberellic acid (GA) biosynthesis pathway, mutations in which can induce a dwarf phenotype in plants and, therefore, reduce mowing frequency, as demonstrated by our dwarf turf varieties developed using conventional mutagenesis techniques. We have recently employed CRISPR/Cas9 technology to create knockout or knockdown mutations in the GA20oxidase1 gene to develop dwarf tall fescue and perennial ryegrass lines. The CRISPR/Cas9 construct used for editing LaGA20ox1 and LpGA20ox1 has ZmUbi and OsU6a promoters controlling the expression of Cas9 and sgRNA, respectively. We have delivered the CRISPR/Cas9 construct into tall fescue and perennial ryegrass via Agrobacterium-mediated transformation. We have observed a 35-50% reduction in plant height compared to the parental wild-type plants. Furthermore, the leaf widths in these mutants are reduced by 30-60%. Some mutants display a smoother leaf texture compared to the wild type. The T2 homozygous mutant progenies will undergo field evaluation for mowing frequency and fertilizer requirements. We anticipate that some of these gene-edited lines should exhibit a significant reduction in mowing frequency and fertilizer input.
Pairwise is a technology-focused food and agriculture company that makes tiny alterations to plant genetics to solve big problems. Powered by our best-in-class technology, Pairwise is harnessing new genomics technologies to create innovative new products. With our FulcrumTM platform, we accelerate innovation in plants and plant-based production systems, delivering value to people and our planet. Partnering with Plant Sciences, Inc. to access their elite germplasm, we created a platform to transform and edit tetraploid blackberry (Rubus sub. Rubus). We used the power of genetics and genomic tools to identify the candidate gene for thornless in blackberries and used CRISPR based gene editing to validate the trait. The identification of this gene will enable faster deployment of a thornless trait, an essential production trait, into blackberries. The gene may be broadly applicable to other Rubus species and horticultural crops such as roses.
Tomato fruit is an important and popular commodity worldwide. One of the main challenges for the fresh market tomato industry is postharvest deterioration, which is mainly determined by the rate of softening. This rate can affect tomato shelf-life, pathogen susceptibility, and fruit waste. The softening rate is regulated by multiple factors, but mainly by the pectin composition of the cell wall, which is remodeled, disassembled, and solubilized by enzymes during fruit ripening; a process that induces and determines the fruit softening in fleshy fruits. The main pectin-degrading enzymes that act on the pectin backbone are Polygalacturonases (PG) and pectate lyases (PL). In this study, we generated a double CRISPR knockout PGPL. We investigated the combined functions of SlPG2a and SlPL on fruit quality traits in postharvest, including shelf-life attributes like firmness and water loss, fruit marketability, and disease incidence. We also assessed additional attributes impacting consumer acceptance, such as taste and aroma. Our findings revealed that the tomato ripening enzymes SlPG2a and SlPL act additively, significantly affecting fruit firmness and shelf-life. Additionally, aspects of fruit quality, such as external color, sugar: acid ratio, and aroma volatiles, were improved or not affected in the double CRISPR knockout PGPL when compared to control. The discoveries of this research provide new insights into the influence of pectin backbone degradation on fruit physiology and postharvest quality, which can be used in crop improvement programs to make fruit more resilient in the supply chain without compromising consumer-based quality traits.
Bacterial wilt, caused by Ralstonia solanacearum (Rs), is an economically devastating plant pathogen that causes rapid death and has been widely distributed worldwide. Rs is a soil-borne bacterium that plugs plant xylem vessels, causing wilt and ultimately death in tomatoes (Solanum lycopersicum L.) and many other economically important crops. The most cost-effective and efficient means of managing Rs is planting resistant cultivars. However, acceptable Rs resistance in these genotypes is tightly linked to small fruit size, preventing development of BW-resistant large fruited tomato cultivars. Previous research has shown that a highly resistant cultivar's candidate resistance gene (Sl-BWR) is linked to qualitative resistance of Rs. Our preliminary data indicates that overexpression of the resistant allele in a susceptible cultivar background confers resistance comparable to the wild-type resistant allele. There is ongoing work to generate susceptible allele overexpression lines and knockout lines from the susceptible and resistant cultivars. We hypothesize the resistant allele knockouts will be susceptible to Rs, indicating that the candidate gene is the primary resistance factor. The gene could be functionally characterized to elucidate the Rs resistance mechanism in tomatoes to be deployed in a breeding program to develop resistant cultivars against bacterial wilt.
Determining the promoter motifs involved in regulating transcription, the first process of gene expression, is critical for synthetic promoter engineering. A gene’s promoter contains cis-regulatory elements, or motifs, which are binding sites for transcription factors (TFs) to initiate and drive transcription. Many bioinformatic tools have been developed for determining statistically overrepresented regions, representing sites of potential cis-regulatory elements, that are shared across groups of promoters. Combining the results produced by multiple bioinformatic tools can lead to improved detection accuracy of motifs conferring biological activity. In the present study, we compiled a set of 11 known soybean constitutive gene promoters under the assumption that some of the promoters are regulated by the same transcription factor(s). Seven bioinformatic tools capable of de novo motif discovery were used to determine potentially shared motifs within the promoters, which were then mapped back to the original promoter sequences. A total of 64 overlapping motif regions (OMRs) were commonly detected amongst the 11 constitutive promoters, and each OMR was cloned individually in front of the minimal CaMV 35S promoter driving GUSPlus reporter gene expression. Transient tobacco leaf agroinfiltration and subsequent quantitative GUS activity assays were used to determine each OMR’s ability to drive reporter gene expression. We found that 20 of the 64 bioinformatically-determined OMRs drove functional gene expression significantly higher than the basal levels conferred by the minimal 35S promoter. Of the 20 functional OMRs, 11 drove GUSPlus expression at levels from just twice that of the minimal 35S promoter up to nearly half of the full-length 35S promoter. We also transformed these 20 functional OMRs individually into Arabidopsis. While GUS staining is still ongoing, we identified a few OMRs which showed strong promoter strength in single-copied homozygous Arabidopsis seedlings. These functional OMRs are strong candidates for further characterization and can be used for crop improvement.
