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 10th. 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 it. The judges were Prakash Kumar from the National University of Singapore, Nagesh Sardesai from Corteva Agriscience™ and Lori Marcum from Corteva Agriscience™. Leyla Hathwaik from USDA-ARS received the first place for her talk “GAANTRY: A precise and robust Agrobacterium-based Gene stacking system for crop improvement”. The second place went to Evelyn Zuniga-Soto for her talk titled “Transcriptomic response of the novel plant engineering bacterium Ensifer adhaerens OV14 during colonization of A.thaliana roots” and the third place went to Nathan T. Reem for his talk on “Application of protoplast technology for genome editing in Physalis species”.

Submitted by Geny Anthony

First Place

GAANTRY: A Precise and Robust Agrobacterium-based Gene Stacking System for Crop Improvement

The genetic engineering of plants provides a means for crop improvement, and the introduction and expression of multiple genes can produce new traits that would otherwise be difficult to obtain through conventional breeding. GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) was shown to be a precise and robust system to stably stack multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA) and obtain high-quality Arabidopsis transgenic events . To examine GAANTRY’s ability to engineer a monocot crop species, a new T-DNA carrying eleven cargo sequences designed for rice transformation was assembled. The 36.8 kilobase pair 11-stack GAANTRY strain allowed the generation transgenic rice. Characterization of 37 independent transgenic events demonstrated that more than 50% of the plants carried all of the inserted cargo. Additionally, 18% of the lines were good-quality events that carried a single copy of the T-DNA free of sequences outside of the T-DNA left border. Therefore, GAANTRY provides a simple, precise and versatile tool for transgene stacking for crop improvement

Leyla Hathwaik, USDA-ARS, Albany, CA. In Vitro Cellular and Developmental Biology, 55:S33, 2019

First Place

GAANTRY: A Precise and Robust Agrobacterium-based Gene Stacking System for Crop Improvement

The genetic engineering of plants provides a means for crop improvement, and the introduction and expression of multiple genes can produce new traits that would otherwise be difficult to obtain through conventional breeding. GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) was shown to be a precise and robust system to stably stack multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA) and obtain high-quality Arabidopsis transgenic events . To examine GAANTRY’s ability to engineer a monocot crop species, a new T-DNA carrying eleven cargo sequences designed for rice transformation was assembled. The 36.8 kilobase pair 11-stack GAANTRY strain allowed the generation transgenic rice. Characterization of 37 independent transgenic events demonstrated that more than 50% of the plants carried all of the inserted cargo. Additionally, 18% of the lines were good-quality events that carried a single copy of the T-DNA free of sequences outside of the T-DNA left border. Therefore, GAANTRY provides a simple, precise and versatile tool for transgene stacking for crop improvement

Leyla Hathwaik, USDA-ARS, Albany, CA. In Vitro Cellular and Developmental Biology, 55:S33, 2019

Second Place

Transcriptomic Response of the Novel Plant Engineering Bacterium Ensifer adhaerens OV14 During Colonization of A. thaliana Roots

Ensifer adhaerens OV14 is a soil-associated alphaproteobacteria with proven ability to transfer T-DNA into plant cells. Since Ensifer Mediated Transformation (EMT) is a viable alternative to Agrobacterium transformation, it is important to understand the basic biology behind the molecular mechanisms conferring DNA transfer capability. A time course experiment involving Arabidopsis thaliana roots colonized by E. adhaerens in the presence of acetosyringone (AS) was set in place. Bacterial RNA was extracted at time points: day 0 (D0), day 1 (D1), day 2 (D2), day 3 (D3), day 5 (D5) and day 7 (D7); T-DNA transfer in root cells was corroborated by histochemical GUS staining, revealing a significant increase in the number of blue foci obtained across time. RNAseq revealed a total of 2333 differentially expressed genes between the root-treated and untreated bacteria. The transcriptomic profile of most of the virulence genes revealed a drastic induction from D0 to D1 followed by a decrease in subsequent time points. The expression profile of chromosomal genes related to quorum sensing, flagellin production and biofilm formation potentially involved in the transformation process were evaluated. These findings suggest a specific transcriptional response occurring in bacterial cells treated with plant tissue. Together, these results are first steps towards the development of projects focused on the improvement of EMT.

Evelyn Zuniga-Soto, Donald Danforth Plant Science Center, St. Louis, MO. In Vitro Cellular and Developmental Biology, 55:S32, 2019

Second Place

Transcriptomic Response of the Novel Plant Engineering Bacterium Ensifer adhaerens OV14 During Colonization of A. thaliana Roots

Ensifer adhaerens OV14 is a soil-associated alphaproteobacteria with proven ability to transfer T-DNA into plant cells. Since Ensifer Mediated Transformation (EMT) is a viable alternative to Agrobacterium transformation, it is important to understand the basic biology behind the molecular mechanisms conferring DNA transfer capability. A time course experiment involving Arabidopsis thaliana roots colonized by E. adhaerens in the presence of acetosyringone (AS) was set in place. Bacterial RNA was extracted at time points: day 0 (D0), day 1 (D1), day 2 (D2), day 3 (D3), day 5 (D5) and day 7 (D7); T-DNA transfer in root cells was corroborated by histochemical GUS staining, revealing a significant increase in the number of blue foci obtained across time. RNAseq revealed a total of 2333 differentially expressed genes between the root-treated and untreated bacteria. The transcriptomic profile of most of the virulence genes revealed a drastic induction from D0 to D1 followed by a decrease in subsequent time points. The expression profile of chromosomal genes related to quorum sensing, flagellin production and biofilm formation potentially involved in the transformation process were evaluated. These findings suggest a specific transcriptional response occurring in bacterial cells treated with plant tissue. Together, these results are first steps towards the development of projects focused on the improvement of EMT.

Evelyn Zuniga-Soto, Donald Danforth Plant Science Center, St. Louis, MO. In Vitro Cellular and Developmental Biology, 55:S32, 2019

Third Place

Application of Protoplast Technology for Genome Editing in Physalis Species

CRISPR/Cas9 has enabled quick and precise generation of mutational gene knockouts. Delivery of CRISPR/Cas 9 editing components into plant cells is routinely conducted by Agrobacterium tumefaciens-mediated transformation. Though reliable, this delivery method has substantial drawbacks: it requires genomic integration of Cas9, and is relatively low-throughput for assessing gene targeting designs for desired outcomes. The drawbacks are compounded when guide RNAs (gRNAs) are inefficient, or with approaches that result in low efficiency, such as promoter modification and gene knock-in. These approaches require higher throughput to generate a sufficient amount of mutagenized alleles. Protoplast transformation is an ideal alternative because high-throughput levels of gene editing can be achieved through transient Cas9 expression. We are developing a protoplast gene editing method for Physalis pruinosa, a semi-domesticated member of the Solanaceae family. First, we optimized protoplast isolation from 2-3-week-old seedlings, and used PEG-mediated transformation for transient expression of a plasmid containing Cas9 and gRNA. Based on this approach, we developed a quick method to screen gRNAs, and obtained accurate estimates of cut efficiency for individual gRNAs. After transformation, protoplasts were cultured in a liquid medium and microcallus formation was observed within 14 days. Microcalli were transferred to solidified medium, and continued dividing to form larger callus within 30 days. We are also pursuing improvement in gene knock-in utilizing small (< 300 bp) homology arms to achieve a 2-codon substitution in the coding sequence of green fluorescent protein, and a single-codon substitution in a native gene homologous to the brix mutation in tomato. Initial results will be presented.

Nathan T. Reem, Boyce Thompson Institute, Ithaca, NY. In Vitro Cellular and Developmental Biology, 55:S32, 2019

Third Place

Application of Protoplast Technology for Genome Editing in Physalis Species

CRISPR/Cas9 has enabled quick and precise generation of mutational gene knockouts. Delivery of CRISPR/Cas 9 editing components into plant cells is routinely conducted by Agrobacterium tumefaciens-mediated transformation. Though reliable, this delivery method has substantial drawbacks: it requires genomic integration of Cas9, and is relatively low-throughput for assessing gene targeting designs for desired outcomes. The drawbacks are compounded when guide RNAs (gRNAs) are inefficient, or with approaches that result in low efficiency, such as promoter modification and gene knock-in. These approaches require higher throughput to generate a sufficient amount of mutagenized alleles. Protoplast transformation is an ideal alternative because high-throughput levels of gene editing can be achieved through transient Cas9 expression. We are developing a protoplast gene editing method for Physalis pruinosa, a semi-domesticated member of the Solanaceae family. First, we optimized protoplast isolation from 2-3-week-old seedlings, and used PEG-mediated transformation for transient expression of a plasmid containing Cas9 and gRNA. Based on this approach, we developed a quick method to screen gRNAs, and obtained accurate estimates of cut efficiency for individual gRNAs. After transformation, protoplasts were cultured in a liquid medium and microcallus formation was observed within 14 days. Microcalli were transferred to solidified medium, and continued dividing to form larger callus within 30 days. We are also pursuing improvement in gene knock-in utilizing small (< 300 bp) homology arms to achieve a 2-codon substitution in the coding sequence of green fluorescent protein, and a single-codon substitution in a native gene homologous to the brix mutation in tomato. Initial results will be presented.

Nathan T. Reem, Boyce Thompson Institute, Ithaca, NY. In Vitro Cellular and Developmental Biology, 55:S32, 2019