To support the Society’s vision to encourage education and scientific informational exchange and recognize outstanding post docs, the Plant Biotechnology Section held a Plant Biotechnology Post-doctoral Oral Presentation Competition on Monday June 8th. A panel of judges evaluated the presentations using the following criteria: experimental design, data analysis, proper interpretation of the results, originality of the study, technical difficulty, appearance and ability of the post-doctoral candidate to present their research. The judges were Fei Zhang (Yale University), Feng Zhang (University of Minnesota), Massimo Bosacchi (KWS), and Alex Conceicao (Calyxt). Ayman Eid from University of Florida received the first place for his talk “Accelerating Optimization of Genome Editing in Sugarcane”. The second place went to Andika Gunadi for his talk titled “Towards Effective Biolistics-mediated Transformation in Hornwort (Anthoceros agrestis) to Unlock Genes Involved in Plant-cyanobacteria Symbiosis”; and the third place went to Hussein Abdullah Ahmed Ahmed for his talk on “Localized Regulation of Hybrid SN19 and GUS Genes Expression Driven by Pathogen Related Promoters in Potato”.

Submitted by Carlos M. Hernandez-Garcia

First Place

Accelerating Optimization of Genome Editing in Sugarcane

Sugarcane (Saccharum spp. hybrid) provides 80% of the world’s sugar and 26% of its bioethanol and is one of the most productive crops due to its superior light conversion, water and nitrogen use efficiencies. Adding additional value streams will be facilitated by metabolic engineering and genome editing of sugarcane. Therefore, sugarcane is a prime candidate feedstock to fuel the emerging bio-economy. Genome editing by sequence specific nucleases (SSN) is revolutionizing crop improvement and is a very promising technology for vegetative propagated polyploid crops of complex genome like sugarcane. Among SSNs, CRISPR\Cas9 and other RNA guided nucleases have been repurposed for genome engineering applications. Despite the flexibility and efficiency of the RNA guided nucleases, several technological challenges remain to achieve specific and efficient multiallelic editing of the highly polyploid sugarcane genome. Therefore, our objectives are to develop a rapid and simple approach to detect CRISPR/Cas9-based alterations in the sugarcane genome and adapt alternative CRISPR/nucleases to sugarcane editing to overcome PAM site limitations. Recombinant DNA vectors were constructed using traditional restriction enzyme cloning and Golden Gate assembly. Single guide RNA’s were confirmed by in vitro cleavage assays prior to biolistic co-transfer with Cas9 nuclease and selectable nptII expression cassettes. Regenerated plants were analyzed phenotypically. Target amplicons were analyzed by restriction enzyme digestion and Sanger sequencing. Targeted mutagenesis of genes involved in chlorophyll biosynthesis resulted in an easily distinguishable phenotype. We will present data describing drop-out frequency caused by co-delivery of two sgRNA’s as well as validation of multi-allelic targeted mutations by Sanger sequencing. Our data confirm a superior approach that efficiently generated distinct phenotypes due to reduced chlorophyll content. This will facilitate the assessment of alternative delivery strategies and RNA guided nucleases to overcome PAM site limitations.

Ayman Eid, DOE Center for Advanced Bioenergy and Bioproducts Innovation, Urbana, IL and University of Florida, Gainesville, FL. In Vitro Cellular and Developmental Biology, 56:S23-24, 2020

First Place

Accelerating Optimization of Genome Editing in Sugarcane

Sugarcane (Saccharum spp. hybrid) provides 80% of the world’s sugar and 26% of its bioethanol and is one of the most productive crops due to its superior light conversion, water and nitrogen use efficiencies. Adding additional value streams will be facilitated by metabolic engineering and genome editing of sugarcane. Therefore, sugarcane is a prime candidate feedstock to fuel the emerging bio-economy. Genome editing by sequence specific nucleases (SSN) is revolutionizing crop improvement and is a very promising technology for vegetative propagated polyploid crops of complex genome like sugarcane. Among SSNs, CRISPR\Cas9 and other RNA guided nucleases have been repurposed for genome engineering applications. Despite the flexibility and efficiency of the RNA guided nucleases, several technological challenges remain to achieve specific and efficient multiallelic editing of the highly polyploid sugarcane genome. Therefore, our objectives are to develop a rapid and simple approach to detect CRISPR/Cas9-based alterations in the sugarcane genome and adapt alternative CRISPR/nucleases to sugarcane editing to overcome PAM site limitations. Recombinant DNA vectors were constructed using traditional restriction enzyme cloning and Golden Gate assembly. Single guide RNA’s were confirmed by in vitro cleavage assays prior to biolistic co-transfer with Cas9 nuclease and selectable nptII expression cassettes. Regenerated plants were analyzed phenotypically. Target amplicons were analyzed by restriction enzyme digestion and Sanger sequencing. Targeted mutagenesis of genes involved in chlorophyll biosynthesis resulted in an easily distinguishable phenotype. We will present data describing drop-out frequency caused by co-delivery of two sgRNA’s as well as validation of multi-allelic targeted mutations by Sanger sequencing. Our data confirm a superior approach that efficiently generated distinct phenotypes due to reduced chlorophyll content. This will facilitate the assessment of alternative delivery strategies and RNA guided nucleases to overcome PAM site limitations.

Ayman Eid, DOE Center for Advanced Bioenergy and Bioproducts Innovation, Urbana, IL and University of Florida, Gainesville, FL. In Vitro Cellular and Developmental Biology, 56:S23-24, 2020

Second Place

Towards Effective Biolistics-mediated Transformation in Hornwort (Anthoceros agrestis) to Unlock Genes Involved in Plant-cyanobacteria Symbiosis

Hornworts (Anthocerophyta) are one of only a few land plants to have evolved the ability for mutualistic symbiosis with cyanobacteria. Unlike other nitrogen-fixing symbionts, cyanobacteria can independently perform photosynthesis, requiring less input from the plant host. Unlocking the genes involved in hornwort-cyanobacteria symbiosis, therefore, holds great potential to improve the productivity of agricultural crops. In vitro methods for the culture of the haploid gametophyte of Anthoceros agrestis with and without cyanobacteria symbionts have been previously developed. Our recent transcriptome analysis of A. agrestis have identified 7 potential genes involved in the symbiotic relationship. However, functional characterization of genes and pathways involved in hornwort-cyanobacteria symbiosis are limited by the absence of reliable transformation and genome editing methodologies. To bridge this gap, we have recently identified critical parameters for boosting the growth of A. agrestis and for effective particle bombardment-mediated transformation. Two weeks after culture, addition of 2% sucrose into the culture medium improved tissue growth by more than two-fold compared to growth on medium without sucrose. Additional parameters, such as green fluorescent protein (GFP) driven by the native hornwort elongation factor 1-alpha (EF1a) promoter, optimized target distance, and tissue culture conditions resulted in greater than 470 GFP-expressing cells at 120 hours post bombardment. Our efforts to further improve transformation and genome editing tools for A. agrestis will pave the way towards unlocking the secrets of plant-cyanobacteria symbiosis. These results are first steps towards the development of projects focused on the improvement of EMT.

Andika Gunadi, Boyce Thompson Institute, Ithaca, NY. In Vitro Cellular and Developmental Biology, 56:S24, 2020

Second Place

Towards Effective Biolistics-mediated Transformation in Hornwort (Anthoceros agrestis) to Unlock Genes Involved in Plant-cyanobacteria Symbiosis

Hornworts (Anthocerophyta) are one of only a few land plants to have evolved the ability for mutualistic symbiosis with cyanobacteria. Unlike other nitrogen-fixing symbionts, cyanobacteria can independently perform photosynthesis, requiring less input from the plant host. Unlocking the genes involved in hornwort-cyanobacteria symbiosis, therefore, holds great potential to improve the productivity of agricultural crops. In vitro methods for the culture of the haploid gametophyte of Anthoceros agrestis with and without cyanobacteria symbionts have been previously developed. Our recent transcriptome analysis of A. agrestis have identified 7 potential genes involved in the symbiotic relationship. However, functional characterization of genes and pathways involved in hornwort-cyanobacteria symbiosis are limited by the absence of reliable transformation and genome editing methodologies. To bridge this gap, we have recently identified critical parameters for boosting the growth of A. agrestis and for effective particle bombardment-mediated transformation. Two weeks after culture, addition of 2% sucrose into the culture medium improved tissue growth by more than two-fold compared to growth on medium without sucrose. Additional parameters, such as green fluorescent protein (GFP) driven by the native hornwort elongation factor 1-alpha (EF1a) promoter, optimized target distance, and tissue culture conditions resulted in greater than 470 GFP-expressing cells at 120 hours post bombardment. Our efforts to further improve transformation and genome editing tools for A. agrestis will pave the way towards unlocking the secrets of plant-cyanobacteria symbiosis. These results are first steps towards the development of projects focused on the improvement of EMT.

Andika Gunadi, Boyce Thompson Institute, Ithaca, NY. In Vitro Cellular and Developmental Biology, 56:S24, 2020

Third Place

Localized Regulation of Hybrid SN19 and GUS Genes Expression Driven by Pathogen Related Promoters in Potato

The present study was focused on localized regulation of hybrid SN19 (cry1Ba-domain I–III and cry1Ia-domain II) and GUS reporter genes driven by pathogen-related AoPR1 promoter isolated from wounded Asparagus officinalis cells, PR1a (pathogenesis-related protein 1a gene promoter) isolated from tobacco and the constitutive CaMV 35S promoter (widely used to express recombinant proteins in plants) on different organs of transgenic potato plants. Potato leaf disc explants and Agrobacterium tumefaciens GV2260 or EHA 105 strains were used in transformation experiments. GUS reporter gene expression driven by AoPR1 and PR1a promoters was specifically localized and increased on wounded sites of leaves, stems and flowers in transgenic tobacco, and leaves, stems, stolons, and micro tubers of transgenic potato plants in both salicylic acid, and wounding treatments. However, GUS gene expression by AoPR1 was undetectable or present in extremely low amounts in non-wounded tissues, in comparison with the levels found in CaMV 35S plants. Moreover, transgenic potato plants carrying a hybrid SN19 gene regulated by AoPR1 promoter showed higher levels of cry gene expression at wounded tissues than CaMV 35S controlled SN19 and exhibited 100% mortality for Leptinotarsa decemlineata and Tuta absoluta Meyrick insects.

Hussein Abdullah Ahmed Ahmed, Ankara University, Ankara, Turkey. In Vitro Cellular and Developmental Biology, 56:S23, 2020

Third Place

Localized Regulation of Hybrid SN19 and GUS Genes Expression Driven by Pathogen Related Promoters in Potato

The present study was focused on localized regulation of hybrid SN19 (cry1Ba-domain I–III and cry1Ia-domain II) and GUS reporter genes driven by pathogen-related AoPR1 promoter isolated from wounded Asparagus officinalis cells, PR1a (pathogenesis-related protein 1a gene promoter) isolated from tobacco and the constitutive CaMV 35S promoter (widely used to express recombinant proteins in plants) on different organs of transgenic potato plants. Potato leaf disc explants and Agrobacterium tumefaciens GV2260 or EHA 105 strains were used in transformation experiments. GUS reporter gene expression driven by AoPR1 and PR1a promoters was specifically localized and increased on wounded sites of leaves, stems and flowers in transgenic tobacco, and leaves, stems, stolons, and micro tubers of transgenic potato plants in both salicylic acid, and wounding treatments. However, GUS gene expression by AoPR1 was undetectable or present in extremely low amounts in non-wounded tissues, in comparison with the levels found in CaMV 35S plants. Moreover, transgenic potato plants carrying a hybrid SN19 gene regulated by AoPR1 promoter showed higher levels of cry gene expression at wounded tissues than CaMV 35S controlled SN19 and exhibited 100% mortality for Leptinotarsa decemlineata and Tuta absoluta Meyrick insects.

Hussein Abdullah Ahmed Ahmed, Ankara University, Ankara, Turkey. In Vitro Cellular and Developmental Biology, 56:S23, 2020