Progress 09/01/08 to 08/31/11
Outputs
OUTPUTS: Experiments were performed to subclone and sequence the termini of linear maize cpDNA molecules. Plastid transformation experiments were continued (from Year 2) using tobacco leaves with either circular or linear transgene vectors. Previously (Year 2), a positive transformant was scored as callus growth on spectinomycin selection medium. In Year 3, selection of positive transformants was based on both spectinomycin and visual GFP expression. Integration of the transgene into the plastid genome also was assessed by PCR amplification. Plastid transformation vectors for maize have been constructed and will now be used for maize plastid transformation experiments. These vectors include plastid-targeting regions corresponding to sequenced termini and GFP (visual) and PMI (metabolic) markers for selection of transformants. Methods were developed to identify and quantify nuclear versions of plastid and mitochondrial sequences (NUPTs and NUMTs) in maize. Information about the extent of cpDNA sequences that have been transferred to the nuclear genome is critical for assessing homoplasmy following transgene integration into the plastid genome. Furthermore, these methods can be easily adapted to other plant species, as well as other organisms and should be useful for phylogenetic analysis, organellar DNA quantification, and clinical testing. The computer program used for genome comparisons is within the public domain (Kumar and Bendich, 2011). The results of our research were presented at the 2011 Meeting of the Society for In Vitro Biology. Undergraduate students assist in this project, either employed as lab assistants or for undergraduate research credits. One student presented her results as a poster for the University of Washington Undergraduate Research Symposium. A tour of our laboratory and description of our research was given to advanced 5th and 6th grade students from the Robinson Center on the UW campus. PARTICIPANTS: PI: Arnold J. Bendich, Professor, Biology, University of Washington. Co-PI: Delene J. Oldenburg, Research Scientist, Biology, University of Washington. Undergraduate student lab assistant: Anna Kovac. General training in lab procedures and research was provided to the undergraduate students and one graduate student, Rachana Kumar. TARGET AUDIENCES: The target audience includes the USDA's National Institute of Food and Agriculture, biologists, botanists, crop scientists, researchers, educators, students, and the general public. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Four distinct terminal sequences were identified. All four correspond to sequences within the inverted repeat region of the plastid genome and are near regions previously identified as end sequences by restriction enzyme analysis. The overall outcome/impact is four pieces of information allowing the design of experimental procedures to use for plastid transformation in maize, a plant that has been recalcitrant to this process. First, knowledge of cpDNA maintenance and degradation identifies the tissue to use for plastid transformation of maize. The base of stalk and dark-grown seedlings retain the "best" cpDNA and thus are the tissues of choice for transformation. Second, plastid transformation of liverwort and tobacco demonstrate that higher transformation efficiency is achieved using a linear vector rather than a circular one. Therefore, a linear vector will be used for maize plastid transformation. Third, the identification of maize cpDNA terminal sequences allows the design of vectors with recombinogenic ends. By using these targeting sequences we expect higher transformation efficiency than with other targeting sequences and circular vectors. Lastly, the information on NUPTs can be used to design PCR primers and introduce unique restriction sites into the plastid vectors, so as to identify true transgene integration into the plastid genome and establish homoplasmy without interference from NUPTs in the nuclear genome.
Publications
- Zheng, Q., Oldenburg, D.J., and Bendich, A.J. (2011) Independent effects of leaf growth and light on the development of the plastid and its DNA content in Zea species. J Exp Botany 62:2715-2730.
- Kumar, R.A. and Bendich A. J. (2011) Distinguishing authentic mitochondrial and plastid DNAs from similar sequences in the nucleus using the polymerase chain reaction. Curr Genet 57:287-295.
- Kovac, A.M., Oldenburg, D.J., and Bendich, A.J. (2011) Transgene integration into the chloroplast genome. University of Washington Undergraduate Research Symposium. May 2011 (Abstract).
- Oldenburg, D.J., Kovac, A.M., and Bendich, A.J. (2011) Plastid transformation: improved efficiency with linear vectors and mechanism of transgene integration. In Vitro Cell Develop Biol (Abstract) 47:S57.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Plastid transformation experiments were conducted using cultured liverworts cells and tobacco leaves with either circular or linear transgene vectors. The efficiency of transformation was also evaluated using young and old tobacco leaves. A positive transformant was scored as callus growth on spectinomycin selection medium. Integration of the transgene into the plastid genome was assessed by PCR amplification and dot blot hybridization. Experiments to assess changes in maize chloroplast DNA (cpDNA) were conducted on light-grown, dark-grown, and dark-to-light transferred maize seedling. The changes in cpDNA content were evaluated using real-time quantitative PCR (qPCR) and blot hybridization. Although our major focus is developing a viable plastid transformation system for maize, the development of these methods may be advanced by insights gained from studying cpDNA maintenance in other plants. For example, the RecA protein may be important in repair of damaged cpDNA and we have evaluated the integrity of cpDNA in recA mutants of Arabidopsis. Undergraduate students assist in this project, either as employed lab assistants or for undergraduate research credits. Two students presented their results as posters for the University of Washington Undergraduate Research Symposium. PARTICIPANTS: PI: Arnold J. Bendich. Co-PI: Delene J. Oldenburg. Undergraduate student lab assistants: Anna Kovac and Joyce Antonio. General training in lab procedures and research was provided to the undergraduate students and one graduate student, Rachana Kumar. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A circular transgene vector is typically used for plastid transformation in plants and, unfortunately, the transformation efficiency is low. Most cpDNA, however, is comprised of linear molecules (monomer, dimer, etc.) and branched concatemers. Thus, we anticipated an improvement in transformation by using linear vectors with recombinogenic ends. We find higher transformation efficiency with a linearized vector than with the circular vector for liverwort cells (Year 1 CRIS report). For initial experiments using tobacco leaves, similar transformations efficiencies were obtained with the circular and linear vectors. We did, however, find a higher number of transformants for the young leaves than with the older leaves. We have begun the process of cloning and sequencing the termini of the linear cpDNA molecules from maize and tobacco. Thus, in Year 3 we anticipate increased efficiency when a linear vector ending in a recombinogenic sequence is used with whole plant tissue. The integrity of the cpDNA may be an important factor for achieving good plastid transformation. Understanding how this property changes during chloroplast/leaf development is therefore important for determining the "best" tissue (young or old, light- or dark-grown) to use for plastid transformation. In our Year 1 CRIS report we noted conflicting results using DAPI fluorescence and real-time qPCR in determining cpDNA content. Additional experiments, including blot hybridization, were performed to resolve this issue. We found a large amount of nupts (nuclear sequences of plastid origin) in maize that may mask changes in authentic cpDNA content and complicate assessment of homoplasmy following transgene integration into the plastid genome. The amount of DNA retained within a chloroplast depends on its maintenance - replication, repair, and degradation - and successful plastid transformation obviously requires "good" cpDNA. Although the mechanism of cpDNA replication has been extensively studied, less is known about repair and degradation. Using the model plant Arabidopsis, we found that cpRecA is necessary for maintaining cpDNA of high integrity. These results suggest differences in RecA expression and/or activity may in part account for the ease or difficulty in achieving plastid transformation in tobacco and maize, respectively.
Publications
- Rowan, B. A. and Bendich, A. J. (2009) The loss of DNA from chloroplasts as leaves mature: fact or artefact J Exp Botany 60:3005-3010.
- Rowan, B. A., Oldenburg, D. J. and Bendich, A. J. (2010) RecA maintains the integrity of chloroplast DNA molecules in Arabidopsis. J Exp Botany 61:2575-2588.
- Kovac, A. M., Oldenburg, D. J., and Bendich, A. J. (2010) Plastid transformation of liverwort cells (abstract), University of Washington Undergraduate Research Symposium. May 2010.
- Antonio, J., Oldenburg, D. J., and Bendich, A. J. (2010) Increasing plastid transformation efficiency in tobacco plants (abstract), University of Washington Undergraduate Research Symposium. May 2010.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Plastid transformation experiments were conducted using cultured liverworts cells with either circular or linear transgene vectors. A positive transformant was scored as a green colony growing on spectinomycin selection medium. Integration of the transgene into the plastid genome was assessed by PCR amplification and dot blot hybridization. Experiments to assess changes in maize chloroplast DNA (cpDNA) were conducted on light-grown, dark-grown, and dark-to-light transferred maize seedling. The changes in cpDNA content were evaluated using DAPI-DNA fluorescence and real-time quantitative PCR (qPCR). Undergraduate students assist in this project either employed as lab assistants or for undergraduate research credits. PARTICIPANTS: Individuals: 1. PI: Arnold J. Bendich, Professor, Biology, University of Washington. 2. Co-PI: Delene J. Oldenburg, Research Scientist, Biology, University of Washington. Training and Professional Development: 1. University of Washington undergraduate student lab assistant: Su Young Yu. 2. General training in lab procedures and research was provided to Su Young Yu and two University of Washington graduate students, Beth Rowan and Jeff Shaver. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A circular transgene vector is typically used for plastid transformation in plants and, unfortunately, the transformation efficiency is low. Most cpDNA, however, is comprised of linear molecules (monomer, dimer, etc.) and branched concatemers. Thus, we anticipated an improvement in transformation by using linear vectors with recombinogenic ends. Indeed, with liverwort cells we did find a higher transformation efficiency (usually 3-10-fold, but 200-fold in one case) with a linearized vector than with the circular vector. Transformation efficiency with cell cultures is generally higher than with whole plant tissue, such as tobacco leaves. Thus, we anticipate increased efficiency when a linear vector is used on whole plant tissue (2nd year). The integrity of the cpDNA may be an important factor for achieving good plastid transformation. Understanding how this property changes during chloroplast/leaf development is therefore important for determining the "best" tissue (young or old, light- or dark-grown) to use for plastid transformation. The cpDNA in maize is retained at a high level in the dark and declines rapidly after transfer in the light as measured by DAPI fluorescence. No change, however, was found by qPCR. Additional experiments are planned to resolve the discrepancy in the results determined by these two methods.
Publications
- Oldenburg, D. J. and Bendich, A. J. 2009. Chloroplasts, Chapter 22 In A. L. Kriz and B. A. Larkins (eds.), Biotechnology in Agriculture and Forestry (Vol 63): Molecular Genetic Approaches to Maize Improvement. Springer-Verlag, Berlin Heidelberg, p. 325-343.
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