Elicitation of Prenylated Stilbenoids in Cell Suspension Cultures of Peanut
Prenylated stilbenoids are inducible defense compounds found in a few plant species such as peanut that have potential applications in human health as anticancer, antiviral and anti-obesity agents. In order to study the biosynthesis of these compounds, cell suspension cultures offer an attractive alternative biological system when compared to the entire plant. To this end, callus cultures were first established from leaf explants obtained from plantlets of peanut of two commercial cultivars, i.e. Andru II and Georgia Green. To induce callus, the leaves were placed in medium containing picloram or benzylamine purine with 2,4-dichlorophenoxyacetic acid. After six weeks, the callus was transferred to liquid medium to start cell suspension cultures. To induce the production of prenylated stilbenoids, the cultures were co-treated with the elicitors methyl jasmonate, cyclodextrin, hydrogen peroxide and magnesium chloride. Prenylated stilbenoids were extracted from the culture medium with ethyl acetate after 192 hours of elicitor treatment and then analyzed via reverse-phase high performance liquid chromatography. The cell suspension cultures of both cultivars secreted into the culture medium the non-prenylated stilbenoid resveratrol and prenylated stilbenoids arachidin-1, arachidin-2, arachidin-3, and arachidin-5. Several other unknown compounds were also induced upon elicitor treatment. This study has shown that cell suspensions cultures of peanut are capable of producing prenylated stilbenoids, and thus these cultures may provide a useful system to study the biosynthesis of these bioactive compounds.
Abbas Karouni, Arkansas Biosciences Institute, Arkansas State University, State University, AR. In Vitro Cellular and Developmental Biology – Plant, 54:488-489, 2018
Identifying Genetic Cues Associated with Enhanced Axonal Growth in Dorsal Root Ganglia Grown Ex Vivo on Nano-scale Grooved Topographical Surfaces
Peripheral nerve injury affects millions of people per year, resulting in potentially chronic nerve damage and a poor quality of life. Current methods to treat peripheral nerve injury vary in recovery outcomes from temporary to permanent sensation loss. Clinical and experimental research aim to improve peripheral nerve regrowth and thus minimize co-morbidities. Peripheral nerve regeneration is a multi-factorial process, requiring cellular and physical cues. Previous research in our laboratory demonstrated that axonal regrowth from mouse dorsal root ganglia (DRG) grown ex vivo can be directed and enhanced when grown on nano-scale grooved topographical surfaces. The purpose of this study is to identify the novel gene(s) responsible for the enhanced and directed axonal growth on nano-scale grooved topography. We selected three genes to analyze based on literature and preliminary proteomic experiments: RhoA, GAP43 and Runx3. Briefly, extracted mouse DRGs were grown ex vivo on nano-scale grooved or control, flat topographical surfaces for six days. Enhanced and directed axonal growth was observed only in the DRGs grown on nano-scale grooved surfaces. RNA was isolated from DRGs and relative expression levels of RhoA, GAP43, and Runx3 were compared using RT-qPCR (n=3). Our data suggests that there is no difference in expression level of RhoA between the DRGs grown on nano-scale grooved surfaces and those grown on flat surfaces. Analysis of the levels of GAP43 and Runx3 are currently being conducted. RNA extracted from identical experiments have been sent for RNAseq analysis which will allow for an unbiased identification of novel genes that are modified in the enhanced and directed growth on the nano-scale grooved surfaces. The results from these studies could be used for developing novel therapeutic modalities aimed at improving peripheral nerve regrowth.
Julie Tamayo, Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 54:S55, 2018
Genetic Transformation of the Ozark Chinquapin (Castanea ozarkensis)
The arrival of the invasive fungal pathogen, Cryphonectria parasitica (cause of the chestnut blight), decimated the native Castanea tree species in the eastern U.S. and southern Ontario, Canada. Billions of American chestnuts and chinquapins were killed. The extirpation of this keystone genus severely impacted the ecology of our forests. Today, the trees persist naturally as stump sprouts, but rarely achieve reproductive maturity. Efforts to restore this genus are critical, as re-sprouting ability is diminishing with time leading to a loss of genetic diversity. Researchers at ESF developed a blight-tolerant American chestnut via genetic engineering by incorporating a common plant defense gene, Oxalate-Oxidase (OxO), into the tree’s genome. OxO detoxifies the oxalic acid produced by the pathogen and inhibits its ability to produce deadly cankers. The objective of this research was to determine if the methodology used to develop a transgenic American chestnut (C. dentata), could be applied to the Ozark chinquapin (C. ozarkensis). Foundational research demonstrated that established methods could be used to initiate somatic Ozark chinquapin embryo cultures and regenerate them into whole plants in-vitro. Somatic embryos were then transformed using established Agrobacterium-mediated transformation methods using an AGL1 strain containing the p35S-OxO binary vector. The presence of the OxO transgene was confirmed in one embryo culture by PCR. It was determined that a transgenic Ozark chinquapin can be developed using the same framework for American chestnut transformation. Continuing research will focus on further optimization of the tissue culture and transformation protocols, determining transgene copy number and expression in the transgenic events, and eventually blight resistance assays. These tools of genetic engineering may help the restoration of the Ozark chinquapin.
Hannah Pilkey, Arkansas Biosciences Institute, State University, AR. In Vitro Cellular and Developmental Biology, 54:S52, 2018