Plant Biotechnology Student Oral Presentation Competition

First Place

Highly Conserved sgRNA Target Sequences Support Cas9-mediated Mutagenesis of LIGULELESS1 in Both Sorghum and Sugarcane

Sugarcane is a prime feedstock for commercial production of biofuel and table sugar. Its highly polyploid genome is the most complex of any domesticated agricultural species (2n = 100–120). This complicates crop improvement by both traditional breeding and genome editing. Sorghum has a diploid genome and its exons have a high level of sequence conservation to those of sugarcane. Establishing protocols to achieve robust, specific, and efficient multiallelic editing remains challenging in most C4 grasses despite the flexibility and efficiency of RNA guided nucleases like CRISPR/Cas9. Therefore, our objectives were to; (1) introduce CRISPR/Cas9-mediated mutations into LIGULELESS1 (LG1) to create a rapidly identifiable phenotype by changing the leaf inclination angle, and (2) evaluate if highly conserved sgRNA target sequences for LG1 support Cas9-mediated mutagenesis in both sorghum and sugarcane. Genome editing reagents were co-delivered into immature embryos of sorghum (var. Tx430; single LG1 copy) and sugarcane calli (var. CP88-1762; 32 LG1 copies) alongside the nptII selectable marker via biolistic gene transfer. The same guide RNAs were used in both crops. Transgenic lines were regenerated following selection, and edits were confirmed via Sanger or NGS sequencing. In sorghum, a single nucleotide, monoallelic insertion at the lg1 target site conferred an upright leaf phenotype in tissue culture that persisted after transfer to soil. T1 progeny of a sorghum event carrying the insertion were analyzed and biallelic lg1 knockouts resulted in a complete lack of ligules and more severe reduction in leaf inclination angle than monoallelic lines. Transgene free, edited events were also recovered following Mendelian segregation. In comparison, sugarcane lines exhibiting between 10-100% lg1 knockout have been obtained and results from phenotypic analysis will be presented. This work highlights lg1 knockout as a suitable strategy for creating a rapidly scorable phenotype and confirms the potential of sorghum as a model species for sugarcane gene editing.

Eleanor Jane Brant, University of Florida, 3054 McCarty Hall, Gainesville, FL. In Vitro Cellular and Developmental Biology, 58:S49-50, 2022

Second Place

Deletion in the GATA Promoter Element of Vacuolar H+ Translocating Pyrophosphatase (V-PPase) by CRISPR/Cas9 Reduces Chalkiness in Rice

Chalkiness is a concern in rice breeding as it drives down the market value of the grain. It is an outcome of improper starch packing that it is induced by heat stress and varies depending on the genotype. In one genotype, chalkiness has been linked to the vacuolar H+ translocating pyrophosphatase (V-PPase), an enzyme that hydrolyzes inorganic pyrophosphate (PPi) as it translocates H+ to the vacuole from the cytoplasm. Natural polymorphisms in the promoter of V-PPase were associated earlier with chalkiness. However, previously described promoters are not present in many genotypes and its mechanism in chalkiness remains unclear. Here, we elucidate the role of V-PPase in chalk formation during grain-filling by (i) comparing its expression across genotypes; (ii) mutagenesis in predicted promoter elements through CRISPR/Cas9 to investigate its impact on chalk size, sucrose content, and granule structure; and (iii) heat perturbation to explore its contribution in regulating cytoplasmic pH. Genotypes with higher chalk were found to have upregulated V-PPase at 10 days after flowering (10DAF). When a high chalk genotype was mutagenized through targeted deletion in the consensus sequence, AGATC, of a GATA promoter element, V-PPase was remarkably downregulated. Coincidingly, chalk size was reduced by approximately 10% under normal temperature and heat stress, granules became slightly more edgy indicating proper starch packing, and sucrose content decreased which suggests its potentially higher utilization for starch biosynthesis. For the heat perturbation in plants with pH sensitive reporters, V-PPase was downregulated and cytoplasmic pH dropped by ~0.21, a condition that could be optimum for enzyme activities essential in grain-filling. Overall, our findings herein illuminate an approach for reducing chalkiness and provide insights into the underlying mechanisms.

Peter James Icalia Gann, University of Arkansas, 125 Plant Sciences Building, Fayetteville, AR. In Vitro Cellular and Developmental Biology, 58:S26, 2022

Third Place

Efficient, Multi-allelic Editing for the Genetic Improvement of Bahiagrass (Paspalum notatum Flüggé)

Polyploidy, apomixis and self-incompatibility complicate the genetic improvement of bahiagrass (Paspalum notatum Flüggé), an important forage and turf grass in the Southeast United States. Gene editing techniques such as CRISPR/Cas9 would bypass chromosome doubling and repeated crosses necessary for conventional breeding of this species and could result in production of improved cultivars in a single generation. A pair of guide RNAs targeting magnesium-protoporphyrin IX chelatase (MgCh) along with Cas9 and the NPTII selectable marker were delivered to embryogenic callus cultures of the apomictic, autotetraploid bahiagrass cultivar ‘Argentine’ using particle bombardment. Chlorophyll-depleted lines, which were confirmed for multi-allelic edits with Sanger and next generation sequencing, were obtained across two independent experiments at efficiencies of 23 and 50% of total transgenic events. This approach has supported further endeavors to genetically improve turf and forage quality in bahiagrass including confirmed targeted mutagenesis of genes involved in functional stay-green, lipid accumulation and lignin composition. In addition, the transmission of edits to apomictic progeny was analyzed, and the feasibility of production of transgene-free edited apomictic lines via heat-inducible Cre/lox site-specific recombination was explored. Overall, these efforts have resulted in an efficient, reproducible CRISPR/Cas9 gene editing protocol for Paspalum notatum, a development with beneficial implications for future breeding efforts in this species.

David May, University of Florida, Agronomy Department, 3062 McCarty Hall D, Gainesville, FL. In Vitro Cellular and Developmental Biology, 58:S25-26, 2022