The following student awards were presented at the 2008 World Congress on In Vitro Biology, Tucson, Arizona. 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 (919) 420-7940, [email protected], or Dr. Pamela Weathers, Chair, Student Affairs and Awards Committee, at [email protected].
2008 WILTON R. EARLE AWARD and SIVB TRAVEL AWARD
Azza H. Mohamed
Use of Serological and Molecular Methods to Determine Citrus Tristeza Virus (CTV) Resistance in Candidate Rootstocks to Replace Sour Orange
Citrus tristeza virus (CTV) is the most economically important virus affecting citrus worldwide. CTV is a phloem limited, single-stranded RNA virus belonging to the Closterovius and vectored by Aphids. The purpose of this study is to screen new citrus rootstock candidates, created in-vitro via protoplast fusion, for citrus tristeza virus (CTV) quickdecline resistance. The hope is to replace sour orange rootstock, the most susceptible rootstock to CTV-induced quick decline. Candidate somatic hybrids rootstocks were screened using a top-working technique by grafting each of 80 selections, mostly mandarin + pummelo somatic hybrids, but also including parental pummelos, along with sour orange onto CTV- infected trees. The virus infection was then detected by serological techniques including tissue blot immunoassay (TBIA), double antibody sandwich enzyme -linked immunosorbent assay (DAS-ELISA) and western analysis. Molecular techniques including multiple molecular markers (MMM) analysis and heteroduplex mobility assay (HMA) followed by the DNA sequencing of the amplified region were applied to detect the differential movement of CTV genotypes into to the test rootstocks. Based on these results, hybrids were classified into 4 different groups based on the different combinations of T30, T36, and VT CTV genotypes that moved into the newly grafted scion material. None of the used serological or molecular methods provided a reliable estimation of virus accumulation. Therefore, quantification of CTV titer was attempted by using reverse transcriptase quantitative real time PCR (qRT-PCR). Based on the preliminary analysis of the qRT-PCR results, some of the tested rootstocks such as A+7-2-99-5 showed a very high quantity of CTV and disease symptoms, making this rootstock very susceptible to CTV infection. On the other hand, rootstocks such as Amb+5-1-99-3, 2247-OP-A2 and pummelo parent HBJL-5 showed very low CTV titer and no disease symptoms, suggesting some resistance to CTV replication and quick decline disease. Many hybrids showed intermediate levels of CTV replication, but no disease symptoms.
Azza H. Mohamed, University of Florida, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850. In Vitro Cellular and Developmental Biology, 44:S59-60, 2008
2008 SIVB TRAVEL AWARD
Luciano Freschi
Oxidative Stress Induces the C3-CAM Transition in Pineapple Plants Grown In Vitro
The switch from C3-photosynthesis to Crassulacean acid metabolism (CAM) in C3-CAM facultative species can be triggered by a variety of environmental factors, such as high light intensity, salinity and drought. A common convergence point of all these abiotic challenges is the generation of reactive oxygen species, which can induce alterations in the cellular redox state. Since changes in the redox balance can activate some plant stress responses, this study attempted to determine if oxidative stress is also involved in the regulation of CAM expression in pineapple plants grown in vitro. To achieve this, micropropagated pineapple plants, all performing C3-photosynthesis, were subjected to CAM induction by water stress and their leaf tissues were analyzed over 30 days for the levels of glutathione (GSH), ascorbate (ASC) and degree of CAM expression. Additionally, the effects of oxidative stress treatments, such as the application of menadione or hydrogen peroxide, were analyzed on the C3-CAM transition. In all cases, the occurrence of C3 or CAM photosynthesis was evaluated by measuring the nocturnal acid accumulation and activities of phosphoenolpyruvate carboxylase (PEPCase) and malate dehydrogenase (MDH). The water stress-induced C3-CAM transition in pineapple was followed by a marked accumulation of GSH and a slight depletion in the ASC pool. Moreover, plants subjected to oxidative stress treatments showed increases in nocturnal acid accumulation and activities of CAM enzymes. The CAM induction by oxidative stress was also accompanied by increases in GSH content and decreases in ASC levels. These results indicate that oxidative stress induces the C3-CAM transition in pineapple, possibly by causing alterations in the cellular antioxidants content and redox status.
Luciano Freschi, University of São Paulo, Department of Botany, Rua José Piragibe, 303, apto. 11, City/St/Zip: São Paulo, São Paulo 05585-040 BRAZIL. In Vitro Cellular and Developmental Biology, 44:S78-79, 2008
2008 CELLULAR TOXICOLOGY AWARD and 2008 HOPE E. HOPPS AWARD
Sumaria Zumarrad
In Vitro Investigation of Antioxidant and Antiproteolytic Properties of the Clove Extracts: Tellimagrandin I and Casuarictin
Neutrophils secrete a variety of substances including Reactive Oxygen species (ROS), Matrix Metalloproteinases (MMP) and Human Neutrophil Elastase (HNE) during inflammation. MMPs and HNE are proteases which degrade elastin, cartilage proteoglycans, several collagens, and fibronectin, as well as foreign materials ingested during phagocytosis. Proteinase inhibitors normally inhibit damage to connective tissue caused by leakage of these enzymes. Insufficient levels of these inhibitors can lead to prolonged inflammation, which is disastrous to human health. Prolonged inflammation can be a major contributor to age related damage, heart disease, diabetes, dental caries, eye disease, arthritis and dementia to name a few. Our lab frequently tests compounds for their antioxidant and antiproteolytic activity so that they can be used as anti-inflammatory agents in topical creams and drugs. Antioxidants and proteinase inhibitors are important to overcome the actions of ROS in vivo. Among the most important dietary antioxidants are plant phenols such as those which can be isolated from tea, rosemary, and spices such as sage, turmeric and clove. We tested the clove extracts: Tellimagrandin I and Casuarictin (Nacalai Tesque Inc, Japan) and both showed significant antioxidant and antiproteolytic activity. Tellimagrandin and Casuarictin are Gallic acid glycosides which are esters of Gallic acid bound to hydroxyl of sugars. Gallic acid is a subclass of polyphenol responsible for the antioxidant activity in many plants. Both extracts were diluted to varying concentrations as low as 0.01ug/mL and assayed separately for antioxidant, anti MMP-8, and anti HNE activity in 96 well plates. Both extracts showed antioxidant and antiproteolytic activity and are readily available, which makes them promising compounds for potential applications in chronic wound therapy, skin care, and oral hygiene.
Sumaria Zumarrad, c/o E. Roemer, Department of Pathology, SUNY Stony Brook, Stony Brook, NY 11794-8691. In Vitro Cellular and Developmental Biology, 44:S47, 2008
2008 HONOR B. FELL AWARD and 2008 SIVB TRAVEL AWARD
Shaneen Baxter
Granulocytic Differentiation of HL-60 Promyelocytic Leukemia Cells is Associated with Increased mRNA Expression for Components of the Cullin-5 Containing E3 Ubiquitin Ligase
The human HL-60 promyelocytic leukemia cell line has been widely used as a model for studying granulocytic differentiation. All-trans retinoic acid (ATRA) treatment of HL-60 cells promotes granulocytic differentiation and is effective as differentiation therapy for patients with acute promyelocytic leukemia. The identification of genes that are transcriptionally regulated by ATRA has provided insight into granulocytic differentiation and differentiation therapy. The Asb-2 gene has previously been identified as a transcriptional target in ATRA-treated HL-60 cells. The ASB-2 protein forms an E3 ubiquitin ligase complex with the proteins Cullin 5, ROC2 and Elongin B and C. The purpose of this study was to determine if there is increased expression of mRNAs for the other components of this E3 ubiquitin ligase complex. To induce granulocytic differentiation, HL-60 cells were treated for five days with ATRA, and differentiation was confirmed by examining superoxide anion production, nuclear morphology, and changes in the expression of CD11b, CD13, and CD15. Quantitative real-time PCR was used to measure ASB-2, Cullin 5, ROC2, and Elongin B and C mRNA expression. Granulocytic differentiation of HL-60 cells was associated with a 23-fold, 1.6-fold, and 1.7-fold statistically significant (t test; p<0.05) increase in mRNA expression for ASB-2, Cullin 5, and ROC2, respectively. No significant change was found in Elongin B and C mRNA expression. Increased expression of multiple components of the Cullin 5-containing E3 ubiquitin ligase complex with ATRA treatment of HL-60 cells indicates that this complex may play an important role in granulocytic differentiation. This research was supported in part by NIH CA122003-01 and Midwestern University.
Shaneen Baxter, Midwestern University, Department of Pharmacology, 555 31st Street, Downers Grove, IL 60515. In Vitro Cellular and Developmental Biology, 44:S43, 2008
