The following student awards were presented at the 2010 In Vitro Biology Meeting, St. Louis, Missouri. 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) 562-0600, sivb@sivb.org, or Dr. Pamela Weathers, Chair, Student Affairs and Awards Committee, at weathers@wpi.edu.
2010 WILTON R. EARLE AWARD AND STUDENT TRAVEL AWARD
Je Hyeong Jung
Isolation, Characterization and RNAi-suppression of COMT and 4CL Genes in Sugarcane
Sugarcane (Saccharum sp. hybrids) is a highly productive C4 grass used as the main source of sugar and more recently to produce ethanol, a renewable transportation fuel. Down-regulation of lignin biosynthesis pathway enzymes is a promising strategy to increase the efficiency of bio-ethanol production from hemicellulosic sugarcane residues. In the lignin pathway, 4-coumarate-CoA ligase (4CL) and Caffeic acid 3-O-methyltransferase (COMT) are key enzymes that catalyze the formation of CoA thiol esters of 4-coumarate and other hydroxycinnamates or the methylation of 5-hydroxyconiferaldehyde to sinapaldehyde, respectively. However, sugarcane has a complex polypoid genome and these genes belong to a large gene family with individual members showing a distinct substrate affinity and specificity. In this study, COMT and 4CL family genes were isolated from the commercially important sugarcane cultivar CP 88-1762 by a combination of cDNA library screening and PCR based approaches. Expression analysis allowed identification of candidate genes with differential expression in different tissues. For further characterization RNAi suppression vectors were generated and stably introduced into immature leaf derived callus of sugarcane cultivar CP 88-1762 by biolistic gene transfer. Transgenic events were confirmed by PCR, and Southern blot analysis and NPTII-ELISA, following regeneration of plants and selection with geneticin for expression of the co-transformed selectable NPTII gene. RNAi suppression of the targeted lignin biosynthetic genes was confirmed by Northern blot analysis. Plants are currently grown to maturity and will be analyzed for lignin content by Klason lignin determination.
Je Hyeong Jung, University of Florida, IFAS, Agronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, Gainesville, FL 32611, USA. Abstract number P-168, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.
2010 SIVB STUDENT TRAVEL AWARD
Emrah Kirdok
Cryopreservation of Sequoia sempervirens by Droplet-freezing Method and Evaluation of Post-thaw Tissue Damage by Histoanatomical Observations
Cryopreservation refers to storage of specimens at ultra low temperatures, where almost all biological reactions in cells are hampered, allowing maintenance of the material for theoratically unlimited periods of time. Among the different methods available, ‘droplet-freezing’ is the recently developed one-step freezing method, repeatedly proven to be very effective in inducing cryotolerance of plant tissues. Present study investigated the effect of droplet-freezing on cryopreservation of apical and basal buds of Sequoia sempervirens, i.e., a threatened germplasm which has not been cryopreserved before, neither by traditional slow cooling nor other one-step freezing methods. The procedure of follows as; cold hardening of in vitro shoot cultures, preculturing of the apical and basal buds, excised from in vitro cultures, on sucrose-rich media, treatment of vitrification solutions (PVS2, PVS3, DMSO, from 15 to 180 min) at 0°C by droplet-freezing approach, storage of explants in liquid nitrogen, thawing, and washing and recovery. Histoanatomical observations of the buds, survived or not to after storage in liquid nitrogen, shed a light to the tissue damage caused by such cryoprotective applications, as well as to the conditions that help the cells maintain their integrity and viability. Indeed, results of the study highlighted the importance of sucrose preculturing step, as only the cells that absorbed sugars in their membranes were viable, while the others were sallow with their cytoplasmic content released to intercellular area, and revealed the possibility to conserve Sequoia germplasm following a careful optimization of each cryoprotective step. To our knowledge, this is the first study to report successful cryopreservation of such valuable germplasm, that is currently being conserved only in biosphere reserves.
Emrah Kirdok, Gebze Yuksek Teknoloji Enstitusu Istanbul Caddesi, no:101 Biyoloji Bolumu, Kocaeli, 41400, Turkey. Abstract number P-091, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.
2010 HONOR B. FELL AWARD AND CELLULAR TOXICOLOGY AWARD
S. Richelle Monaghan
The Opportunities of the Microsporidian Anncaliia Algerae: In Vitro They Seem Unlimited
Microsporidia are intracellular parasites that require a host cell in order to proliferate. Taxonomically, microsporidia encompass over 1,200 species and approximately 150 genera, but are informally categorized according to their economically and clinically relevant hosts. These categories are: insect, mammalian, and fish. Anncaliia algerae (previously Brachiola algerae) is a microsporidian originally identified in insects, and had been considered as an agent to control mosquitoes until it was then discovered to be a pathogen of mammals, including humans. Could fish cells be infected? This research demonstrates that, in vitro at 27°C, A. algerae proliferates in insect, mammalian, and fish cells. Goldfish skin (GFSK-S1), zebra fish (Zeb2J), and fathead minnow testis (FHMT) cell lines were infected with A. algerae spores in addition to the insect and mammalian cell lines, Sf9 and RK-13 respectively. Polar tube extrusion could be seen in these cultures, indicative of spore germination. Development of mature spores was apparent after three days, and proliferation of spores continued for 7 days. Mature spores were visible using phase contrast and fluorescence microscopy following staining with 1 µg/ml of 4’,6-diamidino-2-phenylindole (DAPI). To the best of our knowledge, this is the first demonstration that a microsporidian is capable of infecting all three informal categories of hosts. Although some microsporidian species show restrictions and/or tropisms with respect to hosts, this research suggests that A. algerae can infect any animal cell in vitro, which may mean that it is a parasite of opportunity, capable of infecting any host under the appropriate conditions.
S. Richelle Monaghan, University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada N2L 3G1. Oral Presentation, SIVB Animal Cell Culture Applications Contributed Paper Session, Wednesday, June 9, 11:45 am –12:00 pm, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.
2010 SIVB STUDENT TRAVEL AWARD
Direct Embryogenesis from Immature Leaf Explants Accelerates Genetic Transformation of a Commercially Important Sugarcane Cultivar
Transgenic sugarcane plants with improved agronomic and value added traits have been reported in the past. Future developments are expected to lead to commercial release of transgenic sugarcane and may include its development into a biofactory for high value products. Our strategy to improve the genetic engineering protocol for a commercially important sugarcane cultivar (CP 88-1762) is aiming at reducing the time in tissue culture by eliminating a callus phase. This may positively affect the transformation efficiency and the performance of the transgenic plants. A factorial comparison of alternative auxin sources (1-naphthalenacetic acid; 2,4-dichlorophenoxyacetic acid, 4-amino-3,5,6-trichlorophyridine-2-carboxylic acid, 4-chlorophenoxy acetic acid) and cytokinin concentrations (6-benzylaminopurine at 0.4 or 4.0 µM) during culture initiation from immature leaf roll transverse sections, allowed the identification of a superior culture medium for induction of direct embryogenesis. Biolistic transformation of leaf-roll-disk explants of the commercially important cultivar CP-88-1762 was carried out 5-10 days following culture initiation with two alternative antibiotic resistance genes supporting lethal (nptII) or non-lethal (aadA) selection schemes with geneticin or streptomycin respectively. The optimization of pre-culture time before bombardment, particle size, acceleration pressure, subculture scheme and selection protocol supported a drastic reduction of the tissue culture period compared to a protocol that involves gene transfer to callus. Transgenic plants were confirmed by PCR, ELISA and Southern blot analysis. We will provide details on the optimized protocol that supports producing stably transformed sugarcane plants from cultivar CP 88-1762 in soil within less than 3 months.
Yogesh Taparia, University of Florida, Department of Agronomy, 3062 McCarty Hall D, PO Box 110500, Gainesville, FL 32611, USA. Abstract number P-177, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.
