The following student awards were presented at the 2019 In Vitro Biology Meeting in Tampa, Florida. Information related to the available specific student awards can be found on the SIVB website or by contacting the SIVB Business Office (send email to sivb@sivb.org).

2019 SIVB STUDENT TRAVEL AWARD

Soybean Isolated Microspore Culture: Sustained Cell Divisions and Embryo Formation

To our knowledge, a practical system for producing doubled haploids of soybean (Glycine max) is not available. Our previous research identified potential pyramidal stressors to promote the formation of putative gametic (microspore-derived) calli in soybean anther cultures. These pyramidal stressors were applied to the culture of isolated microspores of genotype IAS-5, including: Pretreatment of donor plants at 10°C day/8°C night for 3 days, followed by 4°C overnight; initial incubation at 11°C in the dark with nitrogen starvation; addition of light at 18°C and 25°C incubation. With minimal adjustments in the culture protocol, such as using 2,4-dichlorophenoxyacetic acid as auxin and 6-benzylaminopurine as cytokinin, 100% of the culture replicates exhibited sustained cell divisions leading to formation of complex structures including embryos. Preliminary flow cytometry results indicated the haploid status of the isolated microspores placed into culture, and the occurrence of spontaneous doubling under certain culture conditions. Embryo development has been documented and compared to the androgenesis model system, Brassica Napus, with many similarities. The culture system described here provides a new platform for the study of soybean androgenesis, and a possible strategy for the production of soybean doubled haploids.

Bretton Hale, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 55:S62, 2019

2019 SIVB STUDENT TRAVEL AWARD

The Role of Clostridium ramosum in Promoting the Development of Obesity And Increasing Lipid Absorption

The constitutive androstane receptor (CAR) is a nuclear hormone receptor which plays a role in the regulation of xenobiotic, glucose, and lipid metabolism. Intriguingly germ-free mice, that are resistant to diet-induced obesity, display upregulated CAR and target gene expression in the liver compared to conventionally-raised mice, suggesting a microbial role in its regulation. Our preliminary results show that CAR knockout (KO) mice fed a high fat diet (HFD) exhibit increased adiposity and altered microbiota composition compared to wild-type mice. Thus, we hypothesized that CAR KO mice fed a HFD and given antibiotic treatment would have a lean phenotype similar to those seen in a germ-free model. Before conducting the proposed study, we sought to determine the most effective antibiotic treatment; one that significantly decreases bacterial load and has a minimal effect, if any, on CAR activation. Therefore, we conducted a study using 30 mice on a HFD or a low-fat diet (LFD) (n = 15, n = 15 respectively) that received: water control (n = 5), rifaximin, (non-absorbable broad-spectrum antibiotic, n = 5) or an antibiotic cocktail consisting of metronidazole, cefoperazone, vancomycin, and neomycin (n = 5) to determine which antibiotic group reduced bacterial load, weight gain, and body fat percentage, and increased cecum size. We also intend to measure mRNA levels of CAR and target genes involved in lipid metabolism. Thus far, we have determined that, regardless of diet, the antibiotic cocktail increased cecum size and decreased body fat percentage compared to the control and rifaximin groups. Therefore, the antibiotic cocktail is expected to be a useful model for determining host-microbe interactions involving CAR and host metabolism.

Iara Cassandra V. Ibay, Midwestern University, Downers Grove IL. In Vitro Cellular and Developmental Biology, 55:S41 2019

2019 HONOR B. FELL AWARD & 2019 SIVB STUDENT TRAVEL AWARD

The Role of Gut Microbes in Regulating the Constitutive Androstane Receptor and Metabolism

The constitutive androstane receptor (CAR) is a nuclear hormone receptor which plays a role in the regulation of xenobiotic, glucose, and lipid metabolism. Intriguingly germ-free mice, that are resistant to diet-induced obesity, display upregulated CAR and target gene expression in the liver compared to conventionally-raised mice, suggesting a microbial role in its regulation. Our preliminary results show that CAR knockout (KO) mice fed a high fat diet (HFD) exhibit increased adiposity and altered microbiota composition compared to wild-type mice. Thus, we hypothesized that CAR KO mice fed a HFD and given antibiotic treatment would have a lean phenotype similar to those seen in a germ-free model. Before conducting the proposed study, we sought to determine the most effective antibiotic treatment; one that significantly decreases bacterial load and has a minimal effect, if any, on CAR activation. Therefore, we conducted a study using 30 mice on a HFD or a low-fat diet (LFD) (n = 15, n = 15 respectively) that received: water control (n = 5), rifaximin, (non-absorbable broad-spectrum antibiotic, n = 5) or an antibiotic cocktail consisting of metronidazole, cefoperazone, vancomycin, and neomycin (n = 5) to determine which antibiotic group reduced bacterial load, weight gain, and body fat percentage, and increased cecum size. We also intend to measure mRNA levels of CAR and target genes involved in lipid metabolism. Thus far, we have determined that, regardless of diet, the antibiotic cocktail increased cecum size and decreased body fat percentage compared to the control and rifaximin groups. Therefore, the antibiotic cocktail is expected to be a useful model for determining host-microbe interactions involving CAR and host metabolism.

Elesa Poteres, Midwestern University, Downers Grove IL. In Vitro Cellular and Developmental Biology, 55:S41, 2019

2019 HOPE E. HOPPS AWARD AND 2019 SIVB STUDENT TRAVEL AWARD

Hydroxyproline-O-glycan Engineering in Tobacco Transient Protein Expression: Fish IL-22 and eGFP as Model Systems

Plant-based recombinant protein production is emerging as a promising approach with significant advantages in cost and safety over other eukaryotic and prokaryotic expression systems. One of the leading plant-based platforms for recombinant protein production is a transient Agrobacteria-mediated expression system in Nicotiana benthamiana. Despite the advantages of plant recombinant proteins, the most important bottleneck that limits the commercialization is the low protein yields. Plants have a unique type of O-glycosylation that has potential to enhance the stability and solubility of recombinant proteins expressed using plant platforms. Specifically target gene sequences are fused with a sequence to code for hydroxyproline-O-glycosylated peptide (HypGP) tags. These tags serve to modify the recombinant expressed protein with protective sugars to improve physicochemical stability during purification as well as employment and delivery of the recombinant protein. Therefore, the overall goal of this project is to understand how these HypGP tags affect protein expression, purification and bioactivity using tobacco transient expression system as a biofactory. We have targeted the expression of two model proteins to characterize this HypGP technology: a very unstable rainbow trout interleukin 22 (IL-22) and a readily-monitored enhanced GFP (eGFP). The “sugar coated” IL-22 expression is significantly enhanced over the untagged protein and can be successfully purified. Data showing bioactivity of HypGP-tagged IL-22 to confirm this tag does not interfere with the function of this cytokine will be presented. To understand the underlying mechanism of HypGP modification process of recombinant expressed proteins, we explored the impact of plant stressors such as drought for increasing expression and recovery of sugar coated eGFP in an effort to optimize the Hyp-O-glycosylation technology for the production of recombinant proteins on plant production platforms.

Cristofer Calvo, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 55:S51, 2019

2019 CELLULAR TOXICOLOGY AWARD

Unraveling the Biosynthesis of Prenylated Stilbenoids in Peanut and Their Anti-inflammatory Activities In Vitro

Stilbenoids are a non-flavonoid class of polyphenols that are important for their potential medicinal applications. Resveratrol is one of the most well-studied stilbenoid and several studies have described its anti-inflammatory, antioxidant and anticancer activities. Prenylated stilbenoids, which include arachidin-1 and arachidin-3, are produced to counteract biotic and abiotic stresses in peanut (Arachis hypogaea). Despite their importance to plant and human health, the biosynthesis of prenylated stilbenoids is still poorly understood. To address this issue, we are using the CRISPR/Cas9 gene editing technique to knockout two stilbenoid-specific prenyltransferase genes in peanut hairy roots. Wild type and knock-out hairy root lines of peanut cv. Tifrunner are being developed. HPLC analyses of the wild-type hairy roots treated with elicitors show the presence of prenylated stilbenoids such as arachidin-1, -2, -3, and -5 in the culture medium. Furthermore, the anti-inflammatory activity of the arachidins is being studied using mammalian cell culture. Our preliminary results suggest that the prenylated stilbenoids are not toxic to the cells. These studies will increase our overall understanding of the biosynthetic pathway of arachidins and carry important translational implications of the arachidins as anti-inflammatory compounds.

Md. Rokib Hasan, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 55:S50, 2019

2019 PHILIP R. WHITE AWARD

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 State University, Jonesboro, AR.  In Vitro Cellular and Developmental Biology, 55:S67, 2019

2019 EXCEPTIONAL PLANT RESEARCH AWARD

In Vitro Propagation of Asclepias spp. for Monarch Butterfly Habitat Restoration 

Asclepias fascicularis is one of the few possible hosts for the Monarch butterfly in California, a plant pollinator being considered for Federal Protection under the Endangered Species Act (U.S. Fish and Wildlife Service). The purpose of this study was to investigate the optimal media formulations for propagating Milkweed plants (Asclepias spp.) in-vitro; the overarching goal was to enhance and restore pollinator habitats. Challenges facing habitat restoration in Southern California include difficulties with traditional propagation methods in Asclepias fascicularis and high contamination levels due to close association with insect activity. Micropropagation, callus proliferation, and seed germination trials were performed with the species A. fascicularis. This study is also precedent to and a resource for protocol development involving plants in the same genus (Asclepias), or family (Apocynaceae). Preliminary results revealed optimal concentrations for active shoot growth from nodal segments, yielding approximately 6 nodes per explant after 4 weeks. The preferred shooting medium was comprised of ½ strength MS salts and vitamins, 3% sucrose, 0.6% agar, 0.25 g/L AC and pH of 5.8. Contamination was less of a problem starting from seed than from wild cuttings. The effect of gibberellin proved beneficial for seed germination. The most efficient germination (66.6%) was observed without cold stratification on a medium comprised of ½ strength MS salts and vitamins, 3% sucrose, 0.6% agar, 5.76 µM GA3, and a pH of 5.8. Callus cultures of A. fascicularis were successfully grown from stem segments on media comprised of ½ strength MS salts and vitamins, 3% sucrose, 0.6% agar, 1.13 µM 2,4-D, and 5.8 pH. Further experiments with organogenesis and somatic embryogenesis may include shoot regeneration and the potential induction of phenotypic variability. The protocols developed here may be useful for other related species such as A. speciosa, A. californica, A. erosa, and A. meadii. A. meadii is of distinct interest due to its rare and endangered status and difficulty with seed propagation.

Angelo Alvarez, Los Angeles Pierce College, Woodland Hills, CA. In Vitro Cellular and Developmental Biology, 55:S35, 2019