This year’s In Vitro Biology meeting featured an 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, Kyle Mohler, Michael Kane, and Maren Arling, were impressed with all of the contestants’ knowledge and preparation. They recognized Bhuvan Pathak from University of Arkansas with the 3rd place award, Lauren Erland from University of Guelph with the 2nd place award, and Xiaoting Wang from Arkansas Biosciences Institute, with the 1st place award. The winners were presented with a certificate and a cash award at the meeting. We encourage all plant biotechnology students to consider this opportunity to develop their presentation skills at future meetings.
Submitted by the Business Office
Plant Cell-derived Growth Factors for Ex Vivo Mass Production of Red Blood Cells
Generation of red blood cells from hematopoietic stem cells (HSCs) used for blood transfusion represents one of the focus in regenerative medicine. Erythropoietic growth factors (eGFs), including stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin (EPO), are vital for proliferation and differentiation of HSCs into mature red blood cells (RBCs) ex vivo. However, massive production of RBCs requires significant quantity and high quality of eGFs, making manufacturing at large scale cost prohibitive. Plant cell culture is proposed to be a promising bioproduction platform for growth factors based on the advantages of, fast growing, no risk of human pathogen, and easy downstream separation and purification. However, low protein productivity is a common bottleneck towards commercialization of this production platform. This bottleneck is addressed by the proprietary “designer HypGP engineering” technology that leverages the plant-specific hydroxyproline(Hyp)-O-glycosylation code to design and engineer novel Hyp-O-glycosylated peptides (HypGP) in plant cells. The engineered HypGP can function as a molecular carrier to dramatically increase the secreted yields of fused proteins in plant cell culture. In this study, eGFs were expressed in tobacco BY-2 cells with a HypGP tag either at N-terminus or C-terminus. The secreted protein yields were determined. The biological activities of the secreted eGFs were assayed for their function in stimulating the proliferation of TF-1 cell line (human erythroblast cells). Our research may provide a novel and promising plant cell-based platform to produce large quantity of eGFs that facilitate the hematopoietic stem cell research and clinical applications.
Xiaoting Wang, Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 54:S30, 2018
Tryptophan Mediates Morphogenesis in St. John’s Wort (Hypericum perforatum L.) Via Interplay Between Auxin- and Indoleamine-dependent and Independent Mechanisms
It has been more than 60 years since Skoog and Miller reported the redirection of plant growth to root production by exposure to auxins in culture media but the biochemical mechanisms that control regeneration are still not completely understood. One challenge with understanding regeneration mechanisms is the potential that applied growth regulators may act indirectly or may be metabolized to active growth regulators. For example, it is possible that the effects attributed to auxin may actually be the result of auxin metabolism to tryptophan or melatonin. We hypothesized that tryptophan is metabolized to auxin, melatonin or serotonin inducing de novo organogenesis in St. John’s wort (Hypericum perforatum L.). Root explants from two germplasm lines of St. John’s wort with altered tryptophan to melatonin metabolism and a wildtype control were incubated with 0 or 10 μM auxin or tryptophan for 0, 24, 48 or 72 h. Incubation with tryptophan increased de novo shoot organogenesis while incubation with auxin led to de novo root regeneration. Auxin-induced roots were short, pale and had significant root hair development vs longer dark brown roots developed on control or tryptophan medium. Quantification of tryptophan, auxin, serotonin and melatonin did not show a ubiquitous response, but levels varied both by line and duration of exposure. Additionally, while tryptophan showed differential effects across the three lines tested, auxin did not show variable growth between the three lines. These results suggest tryptophan mediates plant growth in a tryptophan-specific manner which cannot be solely explained by increasing precursor supply and flux through established plant growth regulator pathways.
Lauren Alexandra Elizabeth Erland, University of Guelph, Guelph, ON, CANADA. In Vitro Cellular and Developmental Biology, 54:S28-29, 2018
Evaluating the Mutation Efficiencies of Two Guide RNA in CRISPR Mediated Transgene Deletion in Rice
The use of the selection marker gene in the plant tissue culture to select the putative transformants reduces the time and effort. After the recovery of transformants, they are no longer needed in the product. Owing to the biosafety and governmental regulations, removal of marker genes is highly desirable. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) is a highly efficient and a cost effective gene editing tool that acts through double stranded break repair process leading to a targeted mutagenesis in the genome. We targeted β-glucuronidase (GUS) gene deletion by dual targeting. The CRISPR construct involved two different guide RNAs (gRNA1 and gRNA2) at two different target sites in the GUS gene. The aim of this study was to evaluate the efficiency of two gRNAs in generating mutations by studying six T0 lines and twenty one T1 lines obtained from three T0 lines. In the T0 lines, no targeting was observed in gRNA spacer-1, while gRNA spacer-2 was mutated either in biallelic or multi- mutations fashion in the five lines. In T1 lines, the overall mutation efficiency for gRNA spacer-1 was 38% and for gRNA spacer-2 was 81%. The mutations observed in gRNA spacer-1 were most likely de novo as Cas9 was inherited in all the T1 studied and their respective T0 were not targeted, while the mutations in gRNA spacer-2 were likely both inherited and de novo as the pattern of mutations were either similar or different than parent. For gRNA spacer-1, 24% T1 plants were monoalleleic, 14% had multiple mutations and 62% had no mutation. For spacer-2, 48% T1 lines were mono-allelic, 9% were either biallelic or multiple mutations and 43% had no mutation. Combining both gRNAs, single base insertions at the target site was most common mutation observed followed by deletion and substitution. Despite the difference in the efficiency of two gRNAs, we were able to isolate the homozygous excision lines which demonstrate that Cas9: gRNA system could be used to generate a marker-free plant.
Bhuvan Pathak, University of Arkansas, Fayetteville, AR. In Vitro Cellular and Developmental Biology, 54:S29, 2018