The following student awards were presented at the 2020 World Congress on In Vitro Biology Virtual Pre-recorded Meeting held from June 6-10, 2020. Information on additional awardees at the 2020 World Congress will be presented in the next issue of the In Vitro Report. Information related to the available specific student awards can be found on the SIVB website or by contacting the SIVB Business Office at sivb@sivb.org.

2020 WILTON R. EARLE AND SIVB STUDENT TRAVEL AWARD

Water Deficit Increases Recombinant Protein Production and Hydroxyproline-O-Glycosylation in Tobacco Transient Expression

Plant-based recombinant protein production is emerging as a promising approach with significant advantages in cost and safety over other 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. To understand the underlying mechanism of HypGP modification process of recombinant expressed proteins, we explored the impact of water deficit on N. benthamiana for increasing expression and recovery of the HypGP-tagged EGFP. We used a non-destructive high throughput plant phenotyping system (HTPP) to assess plant fitness for recombinant protein production under water deficit and normal conditions. We found water deficit for two weeks decreases biomass and leaf water content, but it does not impaired photosynthetic efficiency of N. benthamiana plants. In addition, water deficit increases the in planta accumulation of the two HypGP-tagged EGFP glycoforms in the transient expression system. The HTPP system may provide a valuable tool in monitoring water deficit status in the plants to ensure increased recombinant protein production and enhanced Hyp-O-glycosylation of HypGP-tagged recombinant proteins.

Cristofer Calvo, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. In Vitro Cellular and Developmental Biology, 56:S57-58, 2020

2020 SIVB STUDENT TRAVEL AWARD

Physical Factors Increased Quantity and Quality of Micropropagated Apical Shoots of Cannabis sativa L. in a Repeated Harvest System

Sub-culture in micropropagation is the most labor intensive and costly process for shoot tip culture. Shoot tip culture is a preferred method to propagate clean, vegetative stock plants of Cannabis sativa L.  In-vitro clones of C. sativa ‘US Nursery Cherry 1,’ were micropropagated at four different light intensities (25 – 200 µmol m-2 s-1 PPFD provided by 2 red:1ble LED light) in vented or non-vented vessels in factorial arrangement, to observe shoot tips harvested without sub-culturing for four repeated 2-week cutting cycles. The quality of the micro-cuttings was determined by rooting for 2-weeks in an Oasis® phenolic foam saturated with ½ strength Hoagland’s medium under 200 µmol m-2 s-1 PPFD. The number of cuttings increased over the four cycles, with the most cuttings produced in ventilated boxes at a moderately high (appx 100 µmol m-2 s-1 PPFD) light intensity, and all of the micro-cuttings from optimal light and ventilation rooted ex vitro. Cuttings became smaller (by mass) in the second cutting cycle although, mass could be restored with proper light. Light intensity (appx. 100 µmol m-2 s-1 PPFD) and ventilation increased the quantity of micropropagated shoots over four cycles of repeated harvest and all of the shoots were of good quality to be rooted ex vitro. The labor efficiency of micropropagation by shoot tip culture could be improved by using a multi-cycle cutting process which would allow the same material to be repeatedly cut instead of sub-cultured. Shoot tip production from successive cycles was variable, but improved over successive cycles with the appropriate physical factors, when compared to the standard, once-over system in common use. 

Ryan Murphy, Clemson University, Clemson, SC. In Vitro Cellular and Developmental Biology, 56:S56, 2020

2020 CELLULAR TOXICOLOGY AWARD

Prenylated Stilbenoids as Potential Therapeutic Agents for Triple Negative Breast Cancer

Breast cancer is the most prevalent type of cancer in women worldwide. Triple negative breast cancer (TNBC) is known to be one of the deadliest types since it does not respond to hormonal treatments. Therefore, there is an ongoing search for new treatments to increase survival rates for this disease. The goal of this study is to assess the efficacy of prenylated stilbenoids from peanut as natural products for the prevention and treatment of TNBC. To induce the production of these compounds, peanut hairy root cultures were co-treated with elicitors and then the prenylated stilbenoids were purified via semi-preparative high-performance liquid chromatography from extracts of the culture medium. The cytotoxicity of the prenylated stilbenoids arachidin-1 and arachidin-3 and the non-prenylated stilbenoid resveratrol was studied in TNBC cell lines MDA-MB-231 and MDA-MB-436. Epithelial breast cell line MCF-10A was used as a control. Cytotoxicity and apoptosis were measured by the MTS cell proliferation and Apo-ONE Homogeneous Caspase-3/7 assays, respectively. To further investigate the apoptosis and cell cycle stage, cells were studied using flow cytometry after treatment with each stilbenoid. These studies showed that the prenylated stilbenoids exhibited higher cytotoxicity to the cancer cells than non-prenylated stilbenoid resveratrol and arachidin-3 induces higher level of apoptosis when compared with resveratrol. Furthermore, the increased cytotoxicity correlated with increased levels of the apoptosis markers caspase-3 and caspase-7. This highlights the significance to continue research with prenylated stilbenoids in TNBC. Current studies focus on elucidating the signaling pathways affected by these compounds in TNBC cells in order to advance our understanding of the anticancer mechanisms of these natural products.

Sepideh Mohammadhosseinpour, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 56:S21, 2020

2020 GORDON SATO AND WALLY MCKEEHAN AWARD

Optimization of a Polarized 2D Differentiated Human Colonoid Transwell Model

The colonic epithelial barrier plays a crucial role in preventing permeation of toxins and microbiota while allowing absorption of water and nutrients. Colonic epithelial polarity, consisting of distinct apical and basolateral membrane domains, is crucial for regulating these functions. To develop a model of the human colon in vitro, we aimed to optimize a 2D transwell system of colonic monolayers by assessing media components contributing to proliferation, differentiation and polarization. Colonoids were established from donor resections or biopsies and cultured in Matrigel in media containing Advanced DMEM/F-12, 2mM GlutaMax, 10mM Hepes, N2 supplement, B27 supplement minus vitamin A, 1mM N-Acetyl-L-cysteine, 500nM A8301, 10μM SB202190, 10μM Y27632, 100ng/mL EGF and 50% LWRN conditioned media including 10% FBS. Colonoids were trypsin-dissociated and 200,000 cell aggregates were plated per collagen-IV coated 0.33cm2 transwell insert. Medium was replaced after 24hrs with differentiation medium minus stem cell support factors LWRN, SB202190, and with added 10nM gastrin, 50ng/mL EGF, 50ng/mL noggin and experimental concentrations of A8301 and Y27632. Trans-epithelial electrical resistance was recorded to assess monolayer confluence and barrier function while tight junction protein expression was visualized by immunofluorescence. Optimized methods for generating a polarized and differentiated monolayer included components that support initial cell proliferation: adding Rho Kinase inhibitor 2.5μM Y27632 for the first 48hrs while removing TGFβ inhibitor A8301, and pre-treating colonoids with LWRN selected for high Wnt activity to drive a cystic morphology. These conditions were optimal for creating a reproducibly uniform monolayer that models the colonic epithelial barrier. 

Kateryna Karpoff, The University of Michigan Medical School, Ann Arbor, MI. In Vitro Cellular and Developmental Biology, 56:S20-21, 2020

2020 SIVB STUDENT TRAVEL AWARD

Effect of BAP and TDZ on Direct Shoot Organogenesis in Coconut (Cocos nucifera L.)

Coconut is an important tropical crop grown by more than 11 million subsistence farmers in more than 90 coconut producing countries. However, due to its ageing populations, biotic and abiotic threats, the industry needs a massive 3 billion palm replanting program in the next 5 years to meet the growing demand for coconut products. This activity will be difficult due to the current shortage of planting materials, especially elite cultivars, so a rapid clonal propagation method is urgently needed. Over the past 60 years, clonal propagation in coconut has only been achievable through somatic embryogenesis because coconut shoot tips do not have an apparent capacity to produce vegetative lateral bud outgrowths. However, direct organogenesis, a less focused on plant regeneration pathway for coconut, could be another clonal propagation system for coconut. Direct organogenesis, which eliminates a callus phase, lowers the possibility of somaclonal variation and therefore, offers a more secure plantlet regeneration pathway. In addition, it is more time efficient as compared to somatic embryogenesis. This paper provides insights into the potential use of direct organogenesis as a clonal propagation method for coconut. This is further supported by the documented success of direct shoot regeneration of oil palm, which like coconut, does not propagate vegetatively to produce offshoots and can only have a single shoot per palm (non-branching palm) under natural environmental conditions. In general, plant growth regulators (PGRs) have a significant impact on the rate of direct shoot regeneration depending on the type and concentration of PGRs used. As cytokinins have been shown to have significant effects on promoting the growth and proliferation of axillary or adventitious shoots, their effects on direct shoot organogenesis of coconut is outlined in this paper. The influence of various concentrations of 6-benzylaminopurine (BAP) and thidiazuron (TDZ) on the growth of both coconut shoot tips and young inflorescence tissues is reported.

Eveline Yee Yan Kong, The University of Queensland, Gatton, Queensland, Australia. In Vitro Cellular and Developmental Biology, 56:S28, 2020

2020 PHILIP R. WHITE AWARD

The Philip R. White Award is used to supplement expenses of an individual for continuing education in plant tissue culture techniques. The Award, honoring Philip R. White, an eminent teacher and researcher in plant cell tissue culture techniques, is judged on the student’s ability to demonstrate interest, scholastic achievement, and the need.

I was excited when I first found the information on the Philip R. White Memorial Award, which provided opportunities to students who would like to learn tissue culture. As a fourth year PhD student in plant science, specifically working on tomato tissue culture, the most difficult issue for me is to generate sufficient transgenic materials to answer my biological questions. It takes me at least 6 months to get the first generation of transgenic plants, and even longer to screen and obtain stable transgenic lines. One of my Chinese colleagues sent me a paper about the tomato ‘hairy root’ transformation method, which enables fast and easy generation of transgenic materials using Agrobacterium rhizogenes. So, I applied for the Philip R. White Memorial Award to support my trip to UC Davis (USA) to learn the tomato hairy root transformation technique to enable fast and easy generation of transgenic materials using Agrobacterium rhizogenes. And I appreciate that the Philip R. White Memorial Award give me this opportunity.

 My PhD project is on tomato lateral root development and its control by a key transcription factor, SlMYB93. Previous studies from my host lab showed that MYB93 negatively affected lateral root development in the model plant (Arabidopsis), but nothing is known about MYB93 gene function in crops. So, my PhD research aimed to study the function of MYB93 in tomato by using basic biology, molecular genetics, tissue culture and developmental biology. During my visit to Prof Brady’s lab in the US, I learnt and performed the tomato hairy root transformation protocol to acquire enough transgenic materials, which would be used to analyse SlMYB93 gene expression in space and time, and the location of the MYB93 protein within cells.

 During this research visit, I felt so welcomed in Brady lab. Everyone in the lab was very generous to with their time and help, not only with the transformation protocol, but also more widely with my project. Prof Siobhan Brady encouraged me to give a talk about my PhD project in their lab meeting with people from other two labs. This was the first time that I gave a talk in public outside of the University of Birmingham. Not only did this give me a huge confidence boost, but by interacting with people at different stages of their career, it also helped me to plan my future career in research.