The following student awards were presented at the 2019 In Vitro Biology Meeting in Tampa, Florida. Information on additional awardees at the 2019 Meeting 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 (www.sivb.org) or by contacting the SIVB Business Office at sivb@sivb.org.

2019 WILTON R. EARLE AND SIVB STUDENT TRAVEL AWARD

Grafted Aptenia cordifolia (L. f.) Schwant Leaves as Perfusable Tissue Engineered Scaffolds

Availability of transplantation organs and tissues is a major problem. It is challenging to develop a functional vascular network that allows delivery of nutrients and especially O2. Using an original vasculature, especially microvasculature, of a tissue or organ offers potential. Using animal organs involves decellularization, which retains the extracellular matrix (ECM) scaffold by removing cellular contents with chemicals such as sodium dodecyl sulfate (SDS), bleach, and triton. However, decellularizing mammalian tissue and organs is expensive and they are difficult to source. A low cost, more readily available and consistent source of tissues and organs is required. The plant vascular system is similar to that of mammals, so we aimed to design a plant scaffold model allowing bidirectional liquid flow to support mammalian cell growth. To achieve a bidirectional flow we grafted two Aptenia cordifolia leaves together horizontally appressed to one another with petioles facing opposite directions. The abaxial epidermis of the top leaf and the adaxial epidermis of the bottom leaf were removed with a fine tweezer, wounds soaked in 0.1 mg/L NAA and 1 mg/L BAP for 20 seconds before appressing the wounded sides of each leaf horizontally with petioles facing opposite directions. Leaves were decellularized 2-4 wks after grafting, and structural connections between the leaves were visualized in thin sections using H&E and toluidine blue stains. Red food dye was perfused into the grafted leaves through one petiole of one of the leaves after decellularization to observe flow moving from input petiole into the first and then the second of the grafted leaves then exiting via the output petiole. The graft was subsequently decellularized, lyophilized, and stored. They were later rehydrated and recellularized with GFP labeled human breast cancer line MDA-MB231; survival of the DMA-MB231 cells is measured by DNA content. Results suggest these grafted leaves are able to mimic the mammalian bidirectional vascular system and providing a possible bioscaffold for engineering tissues for transplant.

Yueqing Wang, Worcester Polytechnic Institute, Worcester, MA. In Vitro Cellular and Developmental Biology, 55:S50-51, 2019


2019 SIVB STUDENT TRAVEL AWARD

Evaluating Potential Genotoxicity of Imidacloprid with Fish Cell Lines

Imidacloprid is currently the most widely used insecticide for agricultural crops worldwide. It is a systemic pesticide (taken up by plants and retained within plant tissues) that breaks down slowly in soil and tends to move into water, where it poses risks of concern to aquatic organisms including fish. Literature reports have indicated that imidacloprid is generally non-toxic to fish; however, recent studies indicate the possibility of genotoxic effects in some fish species, thus an in vitro evaluation for genotoxicity using various fish cell lines is being carried out. Preliminary experimentation using the micronucleus assay indicated that imidacloprid at the tested concentrations of 0.1 mg/L, 0.5 mg/L, 1.0 mg/L, and 2.0 mg/L all had statistically significant genotoxic effects on the killifish embryo cell line KFE-5, indicating that at these high concentrations, tested, double stranded DNA breakages may occur. At these concentrations, more binucleated cells had micronuclei, and the mean values of micronuclei were significantly higher than those of the negative control. Our preliminary results confirm other scientific research that has shown that imidacloprid has significant cytotoxic and genotoxic effects on non-target organisms such as fish. Additional research is in progress, which includes performing Comet assays and further micronucleus assays using lower doses and different types of fish cell lines. The effects on cells that may come into direct contact with pesticides are of specific interest, thus cell lines derived from gills, integument, and gastrointestinal cells are currently being evaluated.

Brenna Hay, University of the Fraser Valley, Abbotsford, BC, Canada. In Vitro Cellular and Developmental Biology, 55:S40, 2019


2019 SIVB STUDENT TRAVEL AWARD

Silicon Nanoparticles Mitigate the Adverse Effect of Drought Induced by Polyethylene Glycol of In Vitro Banana Shoots

Water stress is a serious environmental restriction that roughly limits plant growth and development. Banana is one of the world’s most important crops, that is water-loving and vulnerable to drought stress. Silicon occurs mainly as monosilicic acid (H4SiO4) in soil and is known for increasing the ability of plants to tolerate stress. Silicon nanoparticles SiO2-NPs of 20-nm size were applied at three concentrations (50, 100, and 150 mg/l) in combination with polyethylene glycol (PEG) as a water deficit agent for the tissue culture media at three levels (1, 2 and 3%) supplementing Murashige and Skoog medium as necessary for in vitro multiplication of banana. Physiological indices, namely concentration of total chlorophyll and total phenols content, peroxidase (POD), polyphenol oxidase (PPO) enzymes, lipid peroxidation, and growth indicators were measured to investigate the tolerance mechanism of banana under drought stress affected by silicon nanoparticles application. Results showed that SiO2-NPs improved shoots growth and multiplication, and enhanced photosynthesis process. The treated plants exhibited significant increase of phenolic compounds production at the low concentration of 50 ppm and reduction in lipid peroxidation however, peroxidase showed accumulation at the highest level of SiO2 compared to the control. Taken together, the results demonstrated that SiO2-NPs application improved the physiological and metabolic mechanism of the in vitro banana shoots under drought stress.

Lamiaa Mahmoud, Mansoura University, EGYPT and Citrus Research and Education Center, University of Florida, Lake Alfred, FL. In Vitro Cellular and Developmental Biology, 55:S54, 2019


 2019 SIVB STUDENT TRAVEL AWARD

Development of Variegated Lettuce Using CRISPR/Cas9 Technology

The need for gene edited plants to combat issues of growing population rate, extreme weather, and reduced agricultural land availability is more evident than ever as growers are struggling to adapt to the changing environment. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has emerged as a powerful approach for precision breeding to create plants with desirable traits. However, gene editing efficiency of CRISPR/Cas9 in plant relies on the efficacy of generating transgenic plant or tissues with high expression of Cas9 endonuclease. Here, we have constructed a CRISPR-Cas9 vector containing a fused NPT-GFP for high-efficiency gene-editing in annual flowering plants. The high expression of GFP during plant regeneration allowed us to minimize the positional effect on T-DNA expression and preferentially select transgenic seedlings with high expression of Cas9. With this construct, we have targeted the variegation gene LsVar2 in lettuce. Forty independent lines were generated, 27 of which showed strong GFP signals. Six of 27 T0 transgenic lines with GFP signals exhibited variegated leaves. Albino seedlings were observed in segregated progenies of these variegated lines, and sequencing results revealed that homozygous mutations created by Cas9 editing occurred to all these albino seedlings. By contrast, heterozygous mutations were only identified in the plants with variegated leaves and significantly reduced chlorophyll content, and these plants were able to grow and mature normally. Therefore, in addition to confirming the efficiency of the CRISPR-Cas9 vector, our data also provide a proof-of-concept for targeting VAR2 gene in ornamental plants where variegation phenotype is highly favorable. In conclusion, the unique designing of our CRISPR/Cas9 construct allow us to improve gene-editing efficiency and efficiently screen non-TDNA mutants through detecting GFP signals during plant regeneration and progeny segregation.

Chi Nguyen, University of Florida, Apopka, FL. In Vitro Cellular and Developmental Biology, 55:S34-35, 2019


2019 SIVB STUDENT TRAVEL AWARD

Ectopic Expression of a Heterologous Glutaredoxin Enhances Tolerance to Multiple Abiotic Stressors and Grain Yield in Field Grown Maize

Abiotic stress (heat and drought) is an important constraint to corn production, particularly during the reproductive stage, which is the most sensitive and critical for seed set and grain yield. Ectopic expression of the Arabidopsis thaliana glutaredoxin S17 (AtGRXS17) gene in tomato resulted in plants with higher heat and drought tolerance than wild-type plants during vegetative growth. Here, we report that ectopic expression of AtGRXS17 in maize also enhanced tolerance to drought and heat stress during reproductive stages under field conditions. In field tests, AtGRXS17-expressing maize events displayed higher kernel set, resulting in a 6-fold increase in yield in comparison to the non-transgenic counterparts when challenged with heat stress at tasseling through grain-filling. Similarly, AtGRXS17-expressing plants yielded 2-fold and 1.5-fold more grain weight per plant than wild-type when challenged with drought stress in field conditions at the tasseling stage and silking stage, respectively. Our results present a robust and simple strategy for meeting rising yield demands in maize.

Tej Man Tamang, Kansas State University, Manhattan, KS. In Vitro Cellular and Developmental Biology, 55:S66-67, 2019


2019 JOHN S. SONG AWARD

Arabian Primrose Mediated Green Synthesis of Silver Nanoparticles and Evaluation of their Anti-oxidant, Anti-cancer, Anti-microbial and Catalytic Potential

Present study reports single-pot biosynthesis of silver nanoparticles (AgNPs) using aqueous root and leaf extracts of Arabian Primrose [Arnebia hispidissima (Lehm.) A. DC]. Aqueous leaf (LE) and root extracts (RE) showed rapid synthesis within 12 and 6 min, respectively. Characterization of AgNPs was done using UV-Vis spectroscopy, XRD, FESEM, EDAX, TEM, DLS and FTIR. They were face centred cubic crystals with different shapes like sphere, rod, triangle, hexagon and polygon. They had negative zeta potential value confirming their high stability. FT-IR analysis showed presence of phenolics, flavonoids and proteins in the plant extract acting as reducing, capping and stabilizing agents. AgNPs showed high antioxidant activity against DPPH and H2O2 radicals. They exhibited dose-dependent cytotoxicity against cervical cancer cell line, HeLa (RE-AgNPs, IC50 4.44 µg/mL and LE-AgNPs, IC50 7.18 µg/mL) and were non-toxic towards non malignant L20 B cell line. Clonogenic assay was done for the HeLa cells treated with increasing concentrations of AgNPs and a dose dependent decrease in the number of colonies was observed. The synthesized AgNPs also exhibited effective antimicrobial activity against seven human pathogenic strains tested. The silver content in AgNPs suspension was estimated using atomic absorption spectrometry. AgNPs mediated catalytic degradation of structurally different dyes namely, methylene blue, methyl red, eosin yellow, crystal violet, methyl orange, trypan blue and safranine was observed. Thus, it is interpreted that the synthesized AgNPs exhibited antioxidant, anticancer, antimicrobial and catalytic activities so they could have tremendous applications in pharmaceutical, biomedical and biotechnological industries.

Shruti Nindawat, University of Delhi, Delhi, India. In Vitro Cellular and Developmental Biology, 55:S63-64, 2019


 2019 JOSEPH F. MORGAN AWARD AND 2019 SIVB STUDENT TRAVEL AWARD

Analyzing the Effects of Environmental Changes on Fish Cell Lines

Climate change is causing dramatic effects on poikilothermic animals such as fish. In this study we evaluated the effects of higher or lower temperatures from the normal growth temperatures on fish cell lines derived from warm- and cold-water fish species. Preliminary observations indicated changes in cell morphology as well as mitochondrial structure. Using phase contrast and fluorescence microscopy with specific fluorescent markers such as ActinGreen, NucBlue and rhodamine 123, as well as with immunofluorescence for specific proteins, we noticed morphological changes with changing temperatures in the cytoplasm, nucleus and mitochondria of all fish cell lines tested. Most cells became enlarged and mitochondria became elongated with lower temperatures, while warmer temperatures caused shortening of mitochondria and irregular cytoplasmic changes. This occurred for all fish cell lines tested including cold-water derived cell lines such as salmonid cell lines, and warm-water fish derived cell lines such as EelB, GFSk-S1 and KFE-5 from eel, goldfish and killifish respectively. Further research is being conducted to examine the significance of these changes.

Arianne Qanbery, University of the Fraser Valley, Abbotsford, BC, Canada. In Vitro Cellular and Developmental Biology, 55:S42-43, 2019