The following student awards were presented at the 2010 In Vitro Biology Meeting, St. Louis, Missouri. Information on additional awardees at the 2010 Meeting 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.net) or by contacting the SIVB Business Office at (919) 562-0600, [email protected], or Dr. Pamela Weathers, Chair, Student Affairs and Awards Committee, at [email protected].
Milana Trifunovic
2010 SIVB STUDENT TRAVEL AWARD
Genetic Transformation of Centaurium erythraea with AtCKX1 and AtCKX2 Genes Using Agrobacterium tumefaciens
The efficient genetic transformation using Agrobacterium tumefaciens strain was established for Impatiens walleriana plants. Axillary buds isolated from in vitro grown shoots were co-cultivated with A. tumefaciens C581C1 carrying pac1 gene. The transformation efficiency was 1.48 %. The transgenic nature of regenerated shoots was confirmed by PCR and RT-PCR analyses. Comparison of phonotypical traits (shoot length, number of nodes per plant, number of leaves, leaf length, and number of axillary buds) of I. walleriana confirmed lack of significant differences between transformed and control plants. The establishment of an efficient transformation method may facilitate the improvement of ornamental plant in terms of the virus resistance.
Milana Trifunovic, Institute for Biological Research “Sinisa Stankovic”, Bulevar despota Stefana 142, Belgrade 11060, SERBIA. Abstract number P-180, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.
Carlos Hernandez-Garcia
2010 JOHN S. SONG AWARD
Rapid Functional Characterization of Soybean Promoters in Transiently- and Stably-transformed Tissues
Promoters are DNA sequences located upstream of gene coding regions and are largely responsible for controlling gene expression. Discovery and analysis of novel promoters will lead to a better understanding of gene regulation in native contexts and new promoters are useful for the development of transgenic crops. Despite their importance and the high value of soybean, only a few soybean promoters have been characterized. For this research, we have characterized 10 Gmubi (Glycine max Ubiquitin) and 10 GmERF (G. max Ethylene Response Factor) promoters using two distinct validation tools, which were optimized for this research. Promoters from these two distinct families were identified from the soybean genome, PCR-cloned and fused to the gfp gene. For the first validation tool, transient GFP expression was quantified using image analysis following introduction of DNAs into lima bean cotyledons via particle bombardment. For the second validation tool, GFP expression in stably-transformed soybean hairy roots was also evaluated. Soybean cotyledon explants, inoculated with Agrobacterium rhizogenes harboring promoter constructs, showed numerous GFP-positive independent hairy root events 14-20 days after inoculation. GFP quantification in hairy roots revealed considerable variation in GFP expression among different hairy root events but expression was consistent for roots derived from individual events. GFP intensity in hairy roots seemed to be associated with transgene copy number. Strength of promoter activity was generally consistent using transient expression and stable transformation. The most highly expressing soybean promoters were Gmubi3 followed by Gmubi1, Gmubi5, Gmubi9, Gmubi7 and GmERF10.
Carlos Hernandez-Garcia, Department of Horticulture and Crop Science, OARDC/The Ohio State University, 1680 Madison Ave., Williams Hall # 225, Wooster OH 44691, USA. Abstract number P-070, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.
Zachary Raymond King
2010 HOPE E. HOPPS AWARD
Construction and Validation of the Novel pANIC Vector Set by Stable and Transient Expression Using in vitro Cultures of Switchgrass (Panicum virgatum L.) and Rice (Oryza japonica L.)
Switchgrass is a native, perennial, warm season pasture grass, and grows nearly ubiquitously throughout the U.S. The U.S Department of Energy selected switchgrass as a renewable cellulosic biomass crop for the production of ethanol, and as a candidate for genetic improvement via stable transformation, primarily to reduce its recalcitrance to saccharification. Currently, improved monocot vectors are needed. Therefore, 16 Gateway-compatible destination vectors designated “pANIC” were constructed to facilitate more efficient screening and expression of transgenes. The pANIC series allows for the insertion of any DNA sequence of interest, including open reading frames (ORF), and allows overexpression (OE) or knock down (KD) of gene targets, the latter using RNA interference (RNAi). Vectors were designed to facilitate biolistic and Agrobacterium tumefaciens-mediated transformations of monocots. Our technology is driven by three main components: a Gateway cassette for OE or KD of a gene target driven by ZmUbi1, as well a hygromycin B (hph) or basta (bar) plant selection cassette driven by OsAct1 and a visual reporter cassette driven by a novel switchgrass ubiquitin promoter PvUbi1, which drives the expression of either GusPlus™ or a novel red fluorescent protein gene (pporRFP). The vector designated pANIC-7A was successfully used to stably transform rice and switchgrass callus cultures which validated the functionality of the pporRFP and hph cassettes in stable transformation of both species. The functionality of GusPlus™ has also been validated in both species. The pANIC vectors will be used in the future in combination with desired gene candidates to improve biomass feedstocks.
Zachary Raymond King, University of Georgia, Center for Applied Genetics and Technology, Athens, GA 30602-6801, USA. Abstract number P-090, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.
Sherif Sherif
2010 JOSEPH F. MORGAN AWARD AND STUDENT TRAVEL AWARD
JAZ Proteins in Peach Have Different Responses and Characteristics Than Their Orthologs in Arabidopsis
The discovery of JAZ proteins as targets for the F-box-type ubiqutin ligase, Coi1, in Arabidopsis solved a missing link in jasmonate signaling in plants. These proteins are degraded in response to the increased levels of JA in the cell that are usually triggered by the herbivory attack or wounding. Although the link between the degradation of JAZ proteins and the induction of wounding-responsive genes has been demonstrated in Arabidopsis,the role of JAZ proteins in other plant species or their responses to different stimuli have not been investigated yet. In this work, we performed in-silico analysis into the peach EST library which ended up with complete sequences of six genes encoding for JAZ proteins. Peach JAZ proteins have the conserved ZIM and Jas domains that determine the characteristics and functions of these proteins. We studied the response of various JAZ genes to a) Xanthomonaspruni (X.pruni) inoculation, b) leaf wounding and c) JA application in peach (Prunus persica)by quantitative RT-PCR. In spite of their close sequence homology to their counterparts in Arabidopsis, the expression of the six JAZ genes in peach is repressed in response to wounding treatment or JA application. Moreover, the expression of JAZ genes after pathogen infection varied among peach varieties. Only in the susceptible variety, most of the JAZ genes were stimulated within 1 hr of inoculation with X. pruni; however, in both susceptible and resistant varieties gene expression dramatically decreased overtime. These results not only contradict the previous data from Arabidopsi but also they suggest that JAZ proteins might have a different role in peach or for that matter in perennials than herbaceous plants. To test this hypothesis we further over-expressed a member of these proteins (Pp-JAZ1)in Arabidopsis and tobacco in order to detect the inducible degradation of the JAZ proteins byJA application. Although the initial results are in line with our hypothesis, we are continuing further investigations with these transgenic plants to confirm our findings.
Sherif Sherif, University of Guelph, Plant Agricultural Dept., 136-78 College Avenue West, Guielph, ON N1G 4S7, CANADA. Oral Presentation, Biotic Stress I, Wednesday, June 9, 2:35 pm – 2:55 pm, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.
Ergun Kaya
2010 PHILIP R. WHITE AWARD
Development of Efficient Micropropagation and Cryopreservation Protocols for ThymusGermplasm
Thymus is one of the most important genus of the Lamiaceae family, that serves as a natural source of phenolic oils, oleoresins, fresh and dried herbs, used all around the world. However, natural Thymus populations are far from being adequate to support such a great and even growing demand for its products. Interests focusing mainly on few selected chemotypes, lead to the loss of the others in nature, which indeed should be preserved to make available the access to a wide range of genetic diversity to be used as a possible source of natural products. Recently developed biotechnological approaches provide unique alternatives for fast multiplication and long-term maintenance of such germplasm. Among these, ‘in vitro micropropagation’ enables propagation of plants under controlled environmental conditions, while ‘cryopreservation’ make available the cost-effective long-term storage of their germplasm at ultra low temperatures. At such temperature, almost all the biological reactions in cells are hampered, paving the way to store the material for theoratically unlimited periods of time. This study was conducted to develop such protocols for Thymus germplasm. Optimizations for micropropagation involved use of cytokinins, auxins, silver nitrate and gibberellic acid for the induction of multiple shoots. Best shoot proliferation (97%) was obtained when MS medium was supplemented with 1 mg l-1 kinetin and 0.3 mg l-1 GA3. Rooting was easily obtained on MS medium, either hormone-free or supplemented with auxins. Cryopreservation methods applied were (i) PVS2 vitrification, (ii) encapsulation-vitirification, and (iii) droplet-freezing. All three methods showed to be effective to induce cryotolerance of thyme shoot tips, obtained from in vitro propagated plantlets.
Ergun Kaya, Gebze Institute of Technology, Istanbul cad. no: 101, Gebze, Kocaeli 41400, TURKEY. Abstract number P-283, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.
