This year’s In Vitro Biology meeting featured a virtual prerecorded oral presentation competition for Plant Biotechnology Students. Presenters were evaluated on experimental design, data analysis, proper interpretation of the results, originality of the study, technical difficulty, and presentation skills. Our expert panel of judges, Sivamani Elumalai (Syngenta Crop Protection, USA), Jeffrey M. Staub (Plastomics, Inc., USA) and Kan Wang (Iowa State University, USA) were very impressed with all the contestants’ knowledge and preparation. They recognized Tayebeh Kakeshpour (Kansas State University, USA) with the 1st place award, Adrian S. Monthony (University of Guelph, Canada) with the 2nd place award, and Uddhab Karki (Arkansas State University, USA) with the 3rd place award. The winners were presented with a certificate and a cash award. We encourage all plant biotechnology students to consider this as an opportunity to develop their presentation skills at future meetings.


Submitted by Veena Veena

First Place

Tomato (Solanum lycopersicum L.) Class II Glutaredoxin Mutants Generated Via CRISPR/Cas9 System Are Susceptible to Multiple Abiotic Stresses

Global environmental change and rapid population growth make transgenic technologies necessary for crop improvement.  CRISPR/Cas9 system can be precisely designed to generate multiplex genome editing, providing a powerful tool for studying functions of gene families in plants.  Glutaredoxins (GRXs) are low molecular weight oxidoreductases that are involved in oxidative stress responses; however, despite their importance, their function in plants has not been well understood.  In this study, we successfully designed and applied pYLCRISPR/Cas9 multiplex vector system to edit Solanum lycopersicum class II glutaredoxins (SlGRXS14, S15, S16, and S17) and used mutant plants to study physiological functions of knock-out genes.  Our genotyping data showed highly efficient gene editing in T0 plants that were genetically inherited to T1 and T2 generations. Transgene-free single and multiple null mutants in T3 were evaluated under control, heat, drought, chilling, cadmium and short photoperiod stress conditions.  In standard conditions, single and multiple mutants did not show any phenotypic differences compared to wild-type plants.  Unsuccessful attempts to find any single, double, triple or quadruple mutant lines containing S15 null mutants suggests that complete loss of function of S15 is embryonic lethal for tomato.  However, upon exposure to different abiotic stresses, wild-type and mutant plants showed significant phenotypic differences that were easily distinguishable.  Phenotyping data suggested that function of GRX gene family is critical for plant’s survival under abiotic stress conditions, making them attractive targets for crop improvement.

Tayebeh Kakeshpour, Kansas State University, Manhattan, KS. In Vitro Cellular and Developmental Biology, 56:S25-26, 2020

First Place

Tomato (Solanum lycopersicum L.) Class II Glutaredoxin Mutants Generated Via CRISPR/Cas9 System Are Susceptible to Multiple Abiotic Stresses

Global environmental change and rapid population growth make transgenic technologies necessary for crop improvement.  CRISPR/Cas9 system can be precisely designed to generate multiplex genome editing, providing a powerful tool for studying functions of gene families in plants.  Glutaredoxins (GRXs) are low molecular weight oxidoreductases that are involved in oxidative stress responses; however, despite their importance, their function in plants has not been well understood.  In this study, we successfully designed and applied pYLCRISPR/Cas9 multiplex vector system to edit Solanum lycopersicum class II glutaredoxins (SlGRXS14, S15, S16, and S17) and used mutant plants to study physiological functions of knock-out genes.  Our genotyping data showed highly efficient gene editing in T0 plants that were genetically inherited to T1 and T2 generations. Transgene-free single and multiple null mutants in T3 were evaluated under control, heat, drought, chilling, cadmium and short photoperiod stress conditions.  In standard conditions, single and multiple mutants did not show any phenotypic differences compared to wild-type plants.  Unsuccessful attempts to find any single, double, triple or quadruple mutant lines containing S15 null mutants suggests that complete loss of function of S15 is embryonic lethal for tomato.  However, upon exposure to different abiotic stresses, wild-type and mutant plants showed significant phenotypic differences that were easily distinguishable.  Phenotyping data suggested that function of GRX gene family is critical for plant’s survival under abiotic stress conditions, making them attractive targets for crop improvement.

Tayebeh Kakeshpour, Kansas State University, Manhattan, KS. In Vitro Cellular and Developmental Biology, 56:S25-26, 2020

Second Place

Flower Power: A Rapid In Vitro Regeneration Protocol from In Vitro Cannabis sativa Inflorescences

The legalization of recreational cannabis (Cannabis sativa L.) in North America has driven the need for large-scale propagation of disease free, chemically defined clones. Currently, cannabis is propagated using stem cuttings from mother plants, which can occupy up to 15% of commercial production space and are susceptible to pests and diseases. In vitro growth of cannabis allows for rapid clonal propagation of axenic plants for research and germplasm storage, but published methods are few and multiplication rates can be low.  A recent study from our lab explored the regenerative potential of cannabis inflorescences, using greenhouse grown flowers.  Subsequent studies have shown that morphologically normal plants can regenerate from in vitro florets in the absence of plant growth regulators (PGRs).  Since cannabis inflorescences are comprised of compact internodes, they represent a meristem-dense region with a high multiplication potential.  We hypothesized that micropropagation using inflorescences can enhance multiplication rates in C. sativa over existing vegetative methods while providing an alternative approach to culturing day neutral (autoflowering) plants and some photoperiod sensitive genotypes.  We explored the use of PGRs (BAP and TDZ) to enhance the multiplication rate of explants regenerated from individual or groups of florets and assessed the incorporation of inflorescences into alginate-based synthetic seeds. This study provides a framework for the rapid multiplication of clonal explants using C. sativa inflorescences.  Our findings have the potential to facilitate the automation of cannabis micropropagation while providing a roadmap to overcoming.

Adrian S. Monthony, University of Guelph, Guelph, Ontario, CANADA. In Vitro Cellular and Developmental Biology, 56:S25, 2020

Second Place

Flower Power: A Rapid In Vitro Regeneration Protocol from In Vitro Cannabis sativa Inflorescences

The legalization of recreational cannabis (Cannabis sativa L.) in North America has driven the need for large-scale propagation of disease free, chemically defined clones. Currently, cannabis is propagated using stem cuttings from mother plants, which can occupy up to 15% of commercial production space and are susceptible to pests and diseases. In vitro growth of cannabis allows for rapid clonal propagation of axenic plants for research and germplasm storage, but published methods are few and multiplication rates can be low.  A recent study from our lab explored the regenerative potential of cannabis inflorescences, using greenhouse grown flowers.  Subsequent studies have shown that morphologically normal plants can regenerate from in vitro florets in the absence of plant growth regulators (PGRs).  Since cannabis inflorescences are comprised of compact internodes, they represent a meristem-dense region with a high multiplication potential.  We hypothesized that micropropagation using inflorescences can enhance multiplication rates in C. sativa over existing vegetative methods while providing an alternative approach to culturing day neutral (autoflowering) plants and some photoperiod sensitive genotypes.  We explored the use of PGRs (BAP and TDZ) to enhance the multiplication rate of explants regenerated from individual or groups of florets and assessed the incorporation of inflorescences into alginate-based synthetic seeds. This study provides a framework for the rapid multiplication of clonal explants using C. sativa inflorescences.  Our findings have the potential to facilitate the automation of cannabis micropropagation while providing a roadmap to overcoming. 

Adrian S. Monthony, University of Guelph, Guelph, Ontario, CANADA. In Vitro Cellular and Developmental Biology, 56:S25, 2020

Third Place

High Yields Secretion of Human Erythropoietin from Tobacco Cell for Ex Vivo Production of Red Blood Cells

Human blood transfusion is crucial in healthcare, which entirely depends on the human donors. Ex vivo generation of clinically available red blood cells (RBCs) from hematopoietic stem cells (HSCs) offers a promising solution to overcome the challenges associated with current use of donor blood, including shortage of blood supply and risk of transfusion-transmitted infections. However, ex vivo expansion and differentiation of HSCs into RBCs requires large quantities of hematopoietic growth factors/cytokines, particularly erythropoietin (EPO) that is a key cytokine responsible for effective erythropoiesis. High-quality functional EPO is increasingly demanded for fundamental research and clinical applications. Plant cell culture is an emerging alternative bioproduction platform for therapeutic proteins, as it offers advantages in safety, scalability and cost over other eukaryotic and prokaryotic systems. However, low protein productivity and secretion is a common bottleneck preventing the commercialization of this platform. To overcome these bottlenecks, we expressed EPO with a designer peptide tag, termed (SP)20 consisting of 20 tandem repeats of a “Ser-Pro” motif.  This de novo designed tag is expected to direct extensive Oglycosylation on each Pro residue in plant cells and function as a molecular carrier in boosting extracellular secretion and stability of EPO. Tobacco codon-optimized EPO gene with the (SP)20 tag attached at either N-terminal or C-terminal region was stably expressed in tobacco BY-2 cells.  BY-2 cell secreted EPO products were purified with hydrophobic interaction chromatography and Ni+ affinity chromatography for their function assay. The yield of (SP)20-tagged EPO was significantly higher than the EPO without the tag. The in vitro expansion and differentiation of hematopoietic stem cell (CD34+ cells) was established to test the plant cell-produced EPO products. This research develops a new plant cell-based platform for high yield production of EPO, facilitating manufacturing of HSCs-derived RBCs at large scale for clinical applications.

Uddhab Karki, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 56:S26-273, 2020

Third Place

High Yields Secretion of Human Erythropoietin from Tobacco Cell for Ex Vivo Production of Red Blood Cells

Human blood transfusion is crucial in healthcare, which entirely depends on the human donors. Ex vivo generation of clinically available red blood cells (RBCs) from hematopoietic stem cells (HSCs) offers a promising solution to overcome the challenges associated with current use of donor blood, including shortage of blood supply and risk of transfusion-transmitted infections. However, ex vivo expansion and differentiation of HSCs into RBCs requires large quantities of hematopoietic growth factors/cytokines, particularly erythropoietin (EPO) that is a key cytokine responsible for effective erythropoiesis. High-quality functional EPO is increasingly demanded for fundamental research and clinical applications. Plant cell culture is an emerging alternative bioproduction platform for therapeutic proteins, as it offers advantages in safety, scalability and cost over other eukaryotic and prokaryotic systems. However, low protein productivity and secretion is a common bottleneck preventing the commercialization of this platform. To overcome these bottlenecks, we expressed EPO with a designer peptide tag, termed (SP)20 consisting of 20 tandem repeats of a “Ser-Pro” motif.  This de novo designed tag is expected to direct extensive Oglycosylation on each Pro residue in plant cells and function as a molecular carrier in boosting extracellular secretion and stability of EPO. Tobacco codon-optimized EPO gene with the (SP)20 tag attached at either N-terminal or C-terminal region was stably expressed in tobacco BY-2 cells.  BY-2 cell secreted EPO products were purified with hydrophobic interaction chromatography and Ni+ affinity chromatography for their function assay. The yield of (SP)20-tagged EPO was significantly higher than the EPO without the tag. The in vitro expansion and differentiation of hematopoietic stem cell (CD34+ cells) was established to test the plant cell-produced EPO products. This research develops a new plant cell-based platform for high yield production of EPO, facilitating manufacturing of HSCs-derived RBCs at large scale for clinical applications. 

Uddhab Karki, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 56:S26-273, 2020