SIVB hosted The 2012 Plant Biotechnology Student Presentation Competition in beautiful Bellevue on June 4. Outstanding student participation was received this year. Based on the quality of their abstracts, top five student authors were invited to give a talk at this session. Students presented interesting and diverse research topics and fielded challenging questions from the audience. The judges Dr. Todd Jones (Pioneer Hi-bred Intl., USA), Dr. Jyoti Rout (Dow AgroSciences, USA), and Dr. Sylvia Mitchell (University of the West Indies) deliberated in what turned out to be a neck and neck for contenders. Sydney Shaw, an undergraduate student from The Pennsylvania State University was the first place winner. The second place was a tie between Micah Stevens (M.S. student from Purdue University) and Sukalya Poothong (Ph.D. student from Oregon State University). Adjudged competition winners were presented with a certificate and a cash award of $250 for the first place and $100 for the second place at the Plant Biotechnology Section Meeting.
Submitted by Sukhpreet Sandhu
First Place
Sydney Shaw
An Improved Temporary Immersion Bioreactor Design for Plant Tissue Culture Propagation
Temporary immersion bioreactors (TIB) have been used extensively for plant propagation. Nearly all designs have utilized pneumatics (air pressure) to move the culture media either between chambers or between compartments to provide intermittent immersion of the plant tissue. We report here a design that overcomes two disadvantages of typical designs: (1) large volumes of gas use, and (2) a rigid culture vessel. By using a media reservoir that is raised and lowered, the movement of liquid is completely decoupled from gas flow rates. Since the culture vessels are not pressurized, the compartment containing the plant tissue can be constructed from a simple plastic bag. The ability to minimize gas flows provides efficient use of gas mixtures such as elevated oxygen or CO2 to enhance plant tissue culture growth. Liquid flows are observed to induce small but significant suction pressures on the order of several inches of water, which introduces the potential for contamination. This can be overcome by a simple inexpensive manifold design for gas delivery to multiple reactors at very low flow rates. The media reservoir manipulation is accomplished with an inexpensive stepper motor and pulley configuration that includes the ability to provide remote monitoring. Progress on developing this reactor system using seedless watermelon tissue will be presented. Two goals of this work are to achieve superior growth to static agar culture (as demonstrated with a rigid flask TIB operated pneumatically), and photoautotrophic growth in the absence of sugar in the culture media.
Sydney Shaw. The Pennsylvania State University, Department of Chemical Engineering, University Park, PA. In Vitro Cellular and Developmental Biology, 48:S39, 2012
Tied for Second Place
Sukalya Poothong
MS Mineral Nutrients Limit Growth and Development of Micropropagated Red Raspberries
In vitro propagation is important for rapid multiplication of a wide range of nursery crops, including red raspberry. The genetic variation of the many red raspberry cultivars makes it difficult to successfully apply one growth medium for all. Although most cultivars will grow on Murashige and Skoog (1962) medium (MS), some display stunting, hyperhydricity, discoloration, callus, leaf spots, or necrosis. The poor growth symptoms are likely caused by suboptimum concentrations of important mineral salts. This study investigated the effect of five groups of MS mineral salts in a 5-dimensional experimental design. Five red raspberry cultivars were tested with 46 treatments selected from the design sphere of five concentrations each of NH4NO3, KNO3, meso elements (CaCl2, KH2PO4 and MgSO4), minor elements (Zn-Mn-Cu-Co-Mo-B-I), and iron. Shoot cultures were grown for three cycles of 3-week transfers before data was taken. Plant quality, multiplication, shoot length and 5 other parameters were evaluated. Results varied by cultivar for some characteristics, but all cultivars had improved growth or appearance with some treatments compared to MS. Mesos were significantly the most limiting factor associated with changes in plant quality, multiplication and shoot length in all cultivars. Increasing iron and minors significantly decreased growth and multiplication in all cultivars except ‘Willamette’. Nitrogen effects varied with the cultivar. Increasing NH4NO3 and KNO3 decreased shoot number in ‘Canby’, ‘Indian Summer’ and ‘Nootka’ but only KNO3 affected the multiplication in ‘Trailblazer’ and ‘Willamette’. Future experiments will address optimizing mesos and nitrogen ratios to improve media for development of red raspberry micropropagation.
Sukalya Poothong. Department of Horticulture, 4017 ALS, Oregon State University, Corvallis, OR 97331. In Vitro Cellular and Developmental Biology, 48:S38-39, 2012
Tied for Second Place
Micah Stevens
Agrobacterium-mediated Transformation and Regeneration of Pumpkin Ash (Fraxinus profunda) Hypocotyls
Fraxinus profunda is both ecologically and economically important and faces extirpation from attack by the non-native emerald ash borer (EAB). The disjointed native range of pumpkin ash (PA) confined mainly to wetlands, floodplains, or river bottoms of the eastern United States further exacerbates this threat, and PA has been listed as a threatened or endangered species in several states. The EAB larvae feed centripetally under the bark effectively girdling and eventually killing an infested tree. There are no reports of innate resistance in any North American Fraxinus species resulting in the loss of millions of trees. Ash containing genes that impart resistance to the EAB would be of great economic and ecological importance to landowners, forest and conservation managers, as well as to the lumber industry. The objective of this research was to develop a protocol for genetic transformation and regeneration of PA. Hypocotyls isolated from mature seeds were cultured for 3-5 days on Murashige and Skoog (MS) medium with 22.2 µM 6-benzyladenine (BA), 4.5 mM thidiazuron (TDZ), 50 mg L-1 adenine hemisulfate (AS), and 10% coconut water (CW). Hypocotyls were then exposed to Agrobacterium strain EHA105 containing the pq35GR vector, with both a β-glucuronidase (GUS) and neomycin phosphotransferase (nptII) gene. Hypocotyls were transformed in a bacterial suspension with 100 mM acetosyringone, 90 s sonication, and 10 min vacuum-infiltration. After 2-3 days co-culture, hypocotyls were rinsed to remove excess Agrobacterium prior to culture on shoot regeneration medium. Adventitious shoots regenerated on MS medium with 13.3 or 22.2 µM BA, 4.5 mM TDZ, 50 mg L-1 AS, 10% CW, 400 mg L-1 timentin, and 20 mg L-1 kanamycin. Results of a replicated factorial experiment indicated that 400 mg L-1 timentin was optimal for control of Agrobacterium growth, while 20 mg L-1 kanamycin was optimal for shoot regeneration and selection of transformed tissue. The presence of GUS and nptII were confirmed by polymerase chain reaction. This research provides the framework for genetic transformation of PA with a gene specific for EAB resistance.
Micah E. Stevens. Dept. of Forestry and Natural Resources, Hardwood Tree Improvement and Regeneration Center (HTIRC), 715 West State St., West Lafayette, IN 47907. In Vitro Cellular and Developmental Biology, 48:S39-40, 2012
