The following student awards were presented at the 2016 World Congress on In Vitro Biology in San Diego, CA. Information on additional awardees at the 2016 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 (www.sivb.org) or by contacting the SIVB Business Office at firstname.lastname@example.org.
2016 JOHN S. SONG AWARD
Metabolic Engineering of Sugarcane for Hyper-accumulation of Oil in Vegetative Biomass
Metabolic engineering to divert carbon flux from sucrose to oil in a high biomass crop like sugarcane has been proposed as a strategy to boost lipid yields per acre for biodiesel production. The energy content of plant oils in the form of triacylglycerols (TAGs) is two-fold greater compared to carbohydrates. However, vegetative plant tissues do not accumulate oil to a significant amount since fatty acid synthesis in these tissues serves primarily membrane construction, in addition TAGs undergo rapid turnover. Therefore, our objectives include: 1.) increasing fatty acid synthesis by expressing WRI1, a transcription activator of fatty acid biosynthetic genes, 2.) increasing fatty acid synthesis by RNAi suppression of tgd1which is involved in lipid reimport into the plastids from the cytoplasm, 3.) increasing TAG synthesis from diacyl-glycerol and acyl-CoA by over-expression of DGAT1, 4.) optimizing TAG storage by expression of oleosin which prevents the access of lipases to TAG storage compartments.
Constitutive single or multiple gene expression/suppression cassettes were generated and co-delivered with the selectable nptII expression cassette by biolistic gene transfer into sugarcane callus. Plants were regenerated on geneticin containing culture medium and analyzed for presence and expression of target constructs by PCR and RT-PCR, respectively. Quantitative real PCR was performed to study the level of gene expression in transgenic TAG plants in different vegetative tissues. Plants were analyzed for TAG content by GC-MS. Line 327 with high expression of DAGAT, OLE and WRI accumulated TAG to 5.5 % of its leaf dry weight and total lipids to almost 10% of its leaf dry weight. This is equivalent to 275-fold increase of TAG compared with non-transgenic sugarcane. Lines with low TAG accumulation co-expressed either fewer transgenes, or expressed the transgenes at lower levels. This research outcome will add value to the abundant sugarcane post-harvest residues for production of advanced biofuels.
Saroj Parajuli, University of Florida, IFAS, Plant Molecular and Cellular Biology Program, Genetics Institute, Agronomy Department, Gainesville, FL and 2Biosciences Dept., Brookhaven National Lab 463, 50 Bell Ave, Upton, NY 11973. In Vitro Cellular and Developmental Biology, 52:S36, 2016
2016 HOPE E. HOPPS AWARD AND 2016 STUDENT TRAVEL AWARD
Preparing Future Brassica juncea Plant Towards Dry and Saline Areas: Gene Pyramiding of Antioxidant Enzymes.
Reactive oxygen species (ROS) derived from molecular oxygen under biotic and abiotic stress such as salinity, drought, extreme temperatures, heavy metals, UV radiation, atmospheric contamination, nutrient starvation and pathogen attacks, which have deleterious effects on cell metabolism. The antioxidant metabolism protects cells from oxidative damage caused by ROS, such as peroxidation of membrane compounds, polysaccharide degradation, enzyme denaturation and DNA lesions Several enzymes act jointly to maintain redox status homeostasis. The antioxidative defense is initated by SOD, which converts superoxide (O2–) radicals to H2O2. The H2O2 that is also potentially harmful is converted to non-toxic water and monodehydroascorbate by the APX enzyme utilizing ascorbate as the electron donor. In the present study, genes for two different cytosolic antioxidant enzymes, superoxide dismutase (AhCuZnSOD) and ascorbate peroxidase (AhcAPX) isolated from salt tolerant cell lines of Arachis hypogaea were simultaneously over expressed in highly important oilseed crop, Brassica juncea (Indian Mustard) to check the effect on alleviation of abiotic stress. Compared with untransformed control plants, transgenic plants survived under longer period of water deficiency and salinity stress and displayed improved recovery after rehydration. The enhanced levels of antioxidant enzymes in the transgenic plants correlated with higher relative water content, improved photosynthetic efficiency, less electrolyte damage, elevated accumulation of compatible osmolytes, less malondialdehyde as well as ROS accumulation and favorable ionic balance under stress conditions compared to untransformed controls. Our analysis showed that an increase in the activity of antioxidant enzymes can significantly enhance salt and dought tolerance in transgenic plants, suggesting an important role of combined over expression of two antioxidant enzymes.
Neelam Prabha Negi, Jawaharlal Nehru University, School of Life Sciences, New Delhi-110067, INDIA. In Vitro Cellular and Developmental Biology, 52:S65, 2016
2016 SIVB STUDENT TRAVEL AWARD
Susceptibility and Race Specific Wheat Genes Induced by Six Puccinia triticina Races
Puccinia triticina, the casual agent of wheat leaf rust, is a devastating disease that can cause up to 40% yield loss. During fungal infection the host plant recognizes proteins, secreted effectors, and other molecules, which trigger a host defense response. Changes in the pathogen effectors and strong varietal selection pressure are responsible for the rapid development of new rust races. This study aims to understand how leaf rust races utilize wheat throughout infection by identifying wheat genes that are induced by individual races and genes that are induced by many races, which could be potential susceptibility genes. Six leaf rust races were evaluated on a single susceptible variety of wheat at six days post inoculation. RNA was sequenced and 63 wheat genes were identified that showed varying expression in response to the different leaf rust races. Out of the 63 genes, 54 wheat genes were characterized for expression patterns during the first seven days of infection using a time course study and real-time PCR. Race specific gene expression was found in two wheat genes that are affected by race shifts on Lr2A, Lr2C, and Lr17A. Potential susceptibility genes were also identified. Seven genes were selected for further investigation based on expression pattern and proposed function. The expression patterns were confirmed by conducting three biological replicates of real-time PCR data. RNAi was used to silence the seven selected wheat genes to further understand their role in leaf rust infection. Transgenic plants were inoculated and small changes in virulence were observed in two biological replicates. Gene expression data was obtained to show gene knock down in the transgenic plants.
Kerri Neugbauer, Kansas State University, Department of Plant Pathology, 4024 Throckmorton Hall, Manhattan, KS 66506. In Vitro Cellular and Developmental Biology, 52:S35, 2016
2016 SIVB STUDENT TRAVEL AWARD
Strong Cytotoxic Potential and Anti-Proliferative Effect of Nardostachys jatamansi D.C. Herbal Extract on Human Glioblastoma Cell Lines Manifested as Cell Cycle Arrest and Mitotic Catastrophe
Glioblastoma is the primary cause of death due to brain tumours and requires an alternative approach for its management, despite the availability of multimodal treatment therapies (Meher et al., 2001, Taylor, 2010, Pan et al., 2015). Plant based medicines are beneficial as they have a synergistic and mult-itargeted action with minimum/no side effects (Amin et al., 2009, Millimouno et al., 2014). In the present investigation a strong bioefficacy of Nardostachys jatamansi D.C. rhizome extract (NJRE) on human Glioblastoma cell lines U87 MG and U373 MG has been seen. Bioassays conducted with NJRE revealed that the IC50 value as obtained through MTT assay was 33.73±3.5, 30.59±3.4 and 28.39±2.9 µg/mL for U87 MG and 36.9±8.8, 24.5±2.7 and 21.5±5.6 µg/mL for U373 MG after 24 h, 48 h and 72 h, respectively. NJRE at 30 µg/mL was able to induce DNA fragmentation in the cells, indicating apoptosis. Acridine orange and Ethidium bromide differential fluorescence staining revealed that the key process of cell death was apoptosis and 20 µg/mL of NJRE was able to induce early apoptosis, whereas higher doses exhibited complete apoptosis. NJRE also exhibited a cell cycle arrest at G0/G1phase at 60 µg/mL and 80 µg/mL as determined through flow cytometry. DAPI staining of the cells after NJRE treatment exhibited severe DNA damage at 60 µg/mL and 80 µg/mL along with excessive nucleation and mitotic catastrophe at 20 µg/mL and 40 µg/mL. These studies have proved that the plant extract has a strong cyto-toxic potential against Glioblstoma, the response being dose specific and time dependent. Incidentally, the extract was seen to be harmless to the normal cell line HEK. This is our first report proving strong anti-cancerous activity of the Nardostachys jatamansi rhizome extract against Glioblastoma.
Himanshi Kapoor, Medicinal Plant Biotechnology and Applied Research Laboratory, Department of Botany, University of Delhi, Delhi, INDIA. In Vitro Cellular and Developmental Biology, 52:S37, 2016
2016 SIVB STUDENT TRAVEL AWARD
Improvement of Embryogenic Callus Production from Coconut (Cocos nucifera L.) Plumule Explant
Commonly known as the ‘Tree of Life’, coconut (Cocos nucifera L.) is one of the most important palm crops in the world. However, its productivity is affected by age, natural disasters and a number of widespread lethal diseases. There is an urgent need to mass produce healthy seedlings for the replantation of affected areas. To do this, rapid multiplication of coconut by somatic embryogenesis is one such approach and has been attempted in the past few years with some degree of achievement, but as yet has not been optimised. Tissue browning due to the excessive production of phenolics during callogenesis is one of the major bottlenecks preventing optimization. The present study, demonstrates the use of 2-aminoindane-2-phosphonic acid (AIP), a competitive inhibitor of phenylalanine ammonia lyase, to prevent phenol production and increase somatic embryogenic callus production from plumule explants. Following the application of AIP (2 mM) a considerable drop (67.1%) in the total phenolic content was observed together with an increase of embryogenic structures on the callus masses (70.8%). Histological observation revealed that the reduction in phenolics was associated with a maximization of the number of emerging embryogenic structures. In addition, plumule age (1 to 4 weeks after germination culture) was also shown to be an important factor. The highest percentage of embryogenic callus was observed when using plumule explants at 2 weeks old. Primary callus was then thin-sectioned and subcultured to produce secondary friable callus which was more feasible to establish liquid-shake culture. This work provides critical insights for the advancement of coconut somatic embryogenesis through scale-up cell suspension culture.
Quang T. Nguyen, The University of Queensland, School of Agriculture and Food Science, St Lucia QLD 4072, AUSTRALIA and Vietnam National University-HCM, School of Biotechnology, International University, Quarter 6, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, 70000, VIETNAM. In Vitro Cellular and Developmental Biology, 52:S35, 2016