Progress 12/15/09 to 12/14/14
Outputs
Target Audience: Target audiences were scientists within the fields of plant biology, agriculture, and plant reproduction as well as students and teachers to whom we have described the potentialimpact of this research on global agriculture. Changes/Problems: A delay was encountered when one objective, conditional expression, did not lead to expected results. A change in strategy, expression of the native gene in a sexual plant, required additional construct design and transformation experiments. In the end, this approach has provided definitive data for a role of ASGR-BBM in parthenogenesis. What opportunities for training and professional development has the project provided? The PI and co-PI have been engaged in research activities that are internationally recognized. Postdocs, Mookkan Muruganantham and Keun Chae, developed new skills and expanded their knowledge of female reproduction in plants. Two undergraduate students, mentored by Joann Conner, were involved in various aspects of the project that have stimulated their interest in scientific research. How have the results been disseminated to communities of interest? Results have been disseminated primarily by presentations and a manuscript has been submitted for publication in a refereed journal. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The transgenic resources developed through this project have been used to test the role that a novel AP2-domain transcription factor may play in embryogenesis in apomicts. A role for ASGR-BBM in parthenogenesis now has been defined and the potential for this gene to provide a key component for the synthesis of apomixis in sexual crop plants can be explored. The ability to induce parthenogenesis in either reduced or unreduced gametes could have application for generating doubled haploids for breeding and for developing apomictic crops. The ASGR-carrier chromosome of apomictic Pennisetum squamulatum has been characterized at the sequence level by comparison of next-generation sequence data, both transcriptomic and genomic, from an apomictic single-chromosome substitution line with a non-apomictic sister line. A candidate gene for parthenogenesis, ASGR-BABYBOOM, is present in the ASGR and is expressed in apomictic ovules. Transgenic lines of tetraploid pearl millet were generated during the course of this project, with the objective of analyzing the role that ASGR-BABYBOOM plays in apomictic reproduction in this genus. To determine the cellular localization of ASGR-BBM expression (objective 1), transgenic lines have been confirmed to contain a full-length ASGR-BBM promoter-gus gene, and GUS expression was observed to be confined to the egg apparatus in ovules of transgenic plants. This expression pattern is consistent with our hypothesis that ASGR-BBM plays a role in embryogenesis and perhaps parthenogenesis. Transgenic lines to silence BBM through RNAi also have been generated (objective 2) and plants with greatly reduced expression of ASGR-BBM have been identified. Embryo development in plants with reduced expression is impaired such that fewer embryos initiate development in the absence of fertilization. For Objective 3, transgenic lines of tetraploid pearl millet containing a conditional expression system using the AlcR/AlcA ethanol inducible system were recovered. AlcR was constructed to be driven by the rice Actin1 promoter while the AlcA promoter was fused to a GUS reporter gene to identify controlled expression of the system. Regenerated lines showed weak AlcR transcription and no detectable GUS expression under ethanol induction. Analysis of gene expression driven by the rice actin1 promoter in pearl millet suggests that this promoter functions differently in Pennisetum than in rice or maize. Given that Objective 4, to determine the role of ASGR-BBML in embryo development by conditional overexpression in reproductive tissues of sexual and apomictic lines, was contingent on development of the conditional expression system, an alternative objective was pursued. Transformants of sexual, tetraploid pearl millet with the native ASGR-BABYBOOM gene from P. squamulatum have been recovered. Multiple transgenic lines and their transgenic progeny showed embryo development in the absence of fertilization. Offspring include dihaploid and tetraploid progeny. Some of the dihaploid progeny also were transgenic and demonstrated a capacity for parthenogenesis. Offspring of dihaploid progeny either were dihaploid or true haploids. Conditional expression was not necessary to achieve proper spatial and temporal expression of ASGR-BBM to achieve parthenogenesis. Parthenogenesis ranged up to 50% in some transgenic progeny based on observation of cleared ovules, although haploid embryo development to maturity was impaired by lack of endosperm formation. Nevertheless, the large number of parthenogenetic embryos shows good potential for recovery of haploids by embryo rescue.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Conner, J.A., M. Muruganantham, K. Chae, H. Huo, P. Ozias-Akins. 2014. ASGR-BABY BOOM-like (ASGR-BBML) from Apomictic Pennisetum squamulatum Confers Parthenogenesis to Transgenic Pearl Millet. https://pag.confex.com/pag/xxii/webprogram/Paper9139.html
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Ozias-Akins, P., J.A. Conner, M. Muruganantham, K. Chae, H. Huo. 2014. Potential Utility of a Parthenogenesis Gene for Apomixis and Haploid Induction. In Vitro Cell. Develop. Biol. Animal 50:S59.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Ozias-Akins, P., J.A. Conner. 2014. Potential impact of apomixis on crop production and food security. ASA, CSSA, SSSA Intl. Annu. Meet. 2-5 Nov, Long Beach, CA, https://scisoc.confex.com/scisoc/2014am/webprogram/Paper88564.html.
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Progress 12/15/13 to 12/14/14
Outputs
Target Audience: Scientists in the field. Changes/Problems: A delay was encountered when one objective, conditional expression, did not lead to expected results. A change in strategy, expression of the native gene in a sexual plant, required additional construct design and transformation experiments. In the end, this approach has provided definitive data for a role of ASGR-BBM in parthenogenesis. What opportunities for training and professional development has the project provided? The PI and co-PI have been engaged in research activities that are internationally recognized. Postdoc,Keun Chae, developed new skills and expanded hisknowledge of female reproduction in plants. How have the results been disseminated to communities of interest? Results have been disseminated primarily by presentations and a manuscript has been submitted for publication in a refereed journal. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Transformants of sexual, tetraploid pearl millet with the native ASGR-BABYBOOM gene from P. squamulatum have been recovered. Multiple transgenic lines and their transgenic progeny showed embryo development in the absence of fertilization. Offspring include dihaploid and tetraploid progeny. Some of the dihaploid progeny also were transgenic and demonstrated a capacity for parthenogenesis. Offspring of dihaploid progeny either were dihaploid or true haploids. Conditional expression was not necessary to achieve proper spatial and temporal expression of ASGR-BBM to achieve parthenogenesis. Parthenogenesis ranged up to 50% in some transgenic lines based on observation of cleared ovules, although haploid embryo development to maturity was impaired by lack of endosperm formation. Nevertheless, the large number of parthenogenetic embryos shows good potential for recovery of haploids by embryo rescue.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Conner, J.A., M. Muruganantham, K. Chae, H. Huo, P. Ozias-Akins. 2014. ASGR-BABY BOOM-like (ASGR-BBML) from Apomictic Pennisetum squamulatum Confers Parthenogenesis to Transgenic Pearl Millet. https://pag.confex.com/pag/xxii/webprogram/Paper9139.html
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Ozias-Akins, P., J.A. Conner, M. Muruganantham, K. Chae, H. Huo. 2014. Potential Utility of a Parthenogenesis Gene for Apomixis and Haploid Induction. In Vitro Cell. Devel. Biol/. Animal 50:S59.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Ozias-Akins, P., J.A. Conner. 2014. Potential impact of apomixis on crop production and food security. ASA, CSSA, SSSA Intl. Annu. Meet. 2-5 Nov, Long Beach, CA, https://scisoc.confex.com/scisoc/2014am/webprogram/Paper88564.html.
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Progress 12/15/12 to 12/14/13
Outputs
Target Audience: Industry and philanthropic organizations with an interest in the potential for apomixis in commercial and developing country environments. Changes/Problems: A delay was encountered when one objective, conditional expression, did not lead to expected results. A change in strategy, expression of the native gene in a sexual plant, required additional construct design and transformation experiments. In the end, this approach has provided definitive data for a role of ASGR-BBM in parthenogenesis. What opportunities for training and professional development has the project provided? The PI and co-PI have been engaged in research activities that are internationally recognized. Postdoc, Keun Chae, is developing new skills inand expanding his knowledge of female reproduction in plants. Two undergraduate students, mentored by Joann Conner,have been involved in various aspects of the project that have stimulated their interest in scientific research. How have the results been disseminated to communities of interest? Results have been disseminated this year primarily by presentations. What do you plan to do during the next reporting period to accomplish the goals? Goals of the project will be accomplished by completing analysis of transgenic plants and publication of the research.
Impacts
What was accomplished under these goals?
The transgenic resources developed through this project have been used to test the role that a novel AP2-domain transcription factor may play in embryogenesis in apomicts. A role for ASGR-BBM in parthenogenesis now has been defined and the potential for this gene to provide a key component for the synthesis of apomixis in sexual crop plants can be explored. The ability to induce parthenogenesis in either reduced or unreduced gametes could have application for generating doubled haploids for breeding and for developing apomictic crops. The ASGR-carrier chromosome of apomictic Pennisetum squamulatum has been characterized at the sequence level by comparison of next-generation sequence data, both transcriptomic and genomic, from an apomictic single-chromosome substitution line with a non-apomictic sister line. A candidate gene for parthenogenesis, ASGR-BABYBOOM, is present in the ASGR and is expressed in apomictic ovules. Transgenic lines of tetraploid pearl millet were generated during the course of this project, with the objective of analyzing the role that ASGR-BABYBOOM plays in apomictic reproduction in this genus. To determine the cellular localization of ASGR-BBM expression (objective 1), transgenic lines have been confirmed to contain a full-length ASGR-BBM promoter-gus gene, and GUS expression was observed to be confined to the egg apparatus in ovules of transgenic plants. This expression pattern is consistent with our hypothesis that ASGR-BBM plays a role in embryogenesis and perhaps parthenogenesis. Transgenic lines to silence BBM through RNAi also have been generated (objective 2) and plants with greatly reduced expression of ASGR-BBM have been identified. Embryo development in plants with reduced expression is impaired such that fewer embryos initiate development in the absence of fertilization. For Objective 3, transgenic lines of tetraploid pearl millet containing a conditional expression system using the AlcR/AlcA ethanol inducible systemwere recovered. AlcR was constructed to be driven by the rice Actin1 promoter while the AlcA promoter was fused to a GUS reporter gene to identify controlled expression of the system. Regenerated lines showed weak AlcR transcription and no detectable GUS expression under ethanol induction. Analysis of gene expression driven by the rice actin1 promoter in pearl millet suggests that this promoter functions differently in Pennisetum than in rice or maize. Given that Objective 4, to determine the role of ASGR-BBML in embryo development by conditional overexpression in reproductive tissues of sexual and apomictic lines, was contingent on development of the conditional expression system, an alternative objective was pursued.Transformants of sexual, tetraploid pearl millet with the native ASGR-BABYBOOM gene from P. squamulatum have been recovered. Multiple transgenic lines are showing embryo development in the absence of fertilization and offspring include dihaploid and tetraploid progeny. Conditional expression was not necessary to achieve proper spatial and temporal expression of ASGR-BBM.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Koltunow, A.M.G., P. Ozias-Akins, and I. Siddiqi. 2013. Apomixis. In: Seed Genomics. P.W. Becraft, Ed. John Wiley & Sons.
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Progress 12/15/11 to 12/14/12
Outputs
OUTPUTS: Transgenic lines of tetraploid pearl millet have been generated containing several genes, with the objective of analyzing the role that ASGR-BABYBOOM plays in apomictic reproduction in this genus. To determine the cellular localization of ASGR-BBM expression, transgenic lines have been confirmed to contain a full-length ASGR-BBM promoter-gus gene and GUS expression was observed to be confined to the egg apparatus in ovules of transgenic plants. This expression pattern is consistent with our hypothesis that ASGR-BBM plays a role in embryogenesis and perhaps parthenogenesis. Transgenic lines to silence BBM through RNAi also have been generated and plants with greatly reduced expression of ASGR-BBM have been identified. Embryo development in plants with reduced expression is impaired such that fewer embryos develop to maturity and these may be arising primarily through sexual reproduction. Finally transformants with elements of a two-component conditional expression system have been recovered. Conditional is preferable to constitutive expression to alleviate the deleterious effects of ectopic BBM expression on development. However, the rice actin promoter that was selected for one of the conditional expression constructs because of its strong expression in maize embryo sacs does not drive expression in ovules of pearl millet to a detectable level. Further analysis of rice actin-gus transgenics has confirmed that the pattern and strength of expression in pearl millet differs from that published for maize. PARTICIPANTS: Peggy Ozias-Akins, Professor and PI, and Joann A. Conner, Assistant Research Scientist and co-PI are jointly directing the project. Dr. Conner is designing and constructing plasmid vectors. Dr. Mookkan Muruganantham, postdoc, has conducted tissue culture, microprojectile bombardment, plant regeneration, selection, and learned plasmid construction techniques. Dr. Dirk Becker, University of Hamburg, Germany, has collaborated by providing a promoter component for the silencing construct. Ms. Gunawati Gunawan provided technical support for transgenic event analysis. All UGA participants in the project have taken advantage of professional development activities offered through the Plant Center and Institute of Plant Breeding, Genetics, and Genomics, University of Georgia. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The transgenic resources developed through this project are being used to test the role that a novel AP2-domain transcription factor may play in embryogenesis in apomicts. If a clearly defined role is observed, ASGR-BBM would provide a key component for the synthesis of apomixis in sexual crop plants.
Publications
- Fox, T., J.A. Conner, W.W. Hanna, M. Cigan, P. Ozias-Akins. 2012. Mapping genes to the ASGR-carrier chromosome from apomictic Pennisetum squamulatum using heterologous comparative genomic hybridization. https://pag.confex.com/pag/xx/webprogram/Paper1529.html
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Progress 12/15/10 to 12/14/11
Outputs
OUTPUTS: Transgenic lines of tetraploid pearl millet have been generated containing several genes, with the objective of analyzing the role that ASGR-BABYBOOM plays in apomictic reproduction in this genus. To determine the cellular localization of ASGR-BBM expression, transgenic lines have been confirmed to contain a full-length ASGR-BBM promoter-gus gene, although GUS expression was not observed in ovules of primary transgenic plants. A subset of the embryos from crosses between the apomictic line and transgenic lines do show low levels of GUS expression. It is possible that expression from the ASGR-BBM promoter requires additional genes on the ASGR-carrier chromosome or that levels are largely below the threshold for histochemical detection, hypotheses that are being tested. Transgenic lines to silence BBM through RNAi also were obtained and are being evaluated. Finally transformants with elements of a two-component conditional expression system have been developed. Conditional expression is required to alleviate the deleterious effects of constitutive BBM expression on development. PARTICIPANTS: Peggy Ozias-Akins, Professor and PI, and Joann A. Conner, Assistant Research Scientist and co-PI are jointly directing the project. Dr. Conner is designing and constructing plasmid vectors. Dr. Mookkan Muruganantham, postdoc, is conducting tissue culture, microprojectile bombardment, plant regeneration, selection, and learning plasmid construction techniques. Dr. Dirk Becker, University of Hamburg, Germany, has collaborated by providing a promoter component for the silencing construct. Ms. Gunawati Gunawan is providing technical support for transgenic event analysis. All UGA participants in the project have taken advantage of professional development activities offered through the Plant Center and Plant Breeding, Genetics, and Genomics Institute, University of Georgia. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The transgenic resources developed through this project are being used to test the role that a novel AP2-domain transcription factor may play in embryogenesis in apomicts. If a clearly defined role is observed, ASGR-BBM would provide a key component for the synthesis of apomixis in sexual crop plants.
Publications
- Conner, J.A., Y. Zeng, H. Huo, E. Albertini, W.W. Hanna, P. Ozias-Akins. 2010. Identification and characterization of ASGR-BABY BOOM-like (ASGR-BBML), a candidate apomixis gene identified in Pennisetum squamulatum and Cenchrus ciliaris. http://www.intl-pag.org/18/abstracts/W06_PAGXVIII_034.html
- Conner, J.A., H. Huo, M. Mookkan, and P. Ozias-Akins. 2011. ASGR-BABY BOOM-like (ASGR-BBML), a candidate apomixis gene for parthenogenesis identified in Pennisetum and Cenchrus species. In Vitro Cell. Develop. Biol.- Animal 47:S39
- Ozias-Akins, P. and J.A. Conner. 2011. Regulation of Apomixis. In: Plant Biotechnology and Agriculture: Prospects for the 21st Century. A. Altman and P.M. Hasegawa, Eds. Elsevier, Amsterdam, Chapter 16
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Progress 12/15/09 to 12/14/10
Outputs
OUTPUTS: The project did not get fully underway until a postdoc with plant tissue culture experience came on board in June 2010. Plant meterials were grown and immature embryos were harvested at regular intervals to establish tissue cultures of tetraploid pearl millet. Simultaneously, ASGR-BBML promoter:uidA constructs were built, either with or without an upstream LTR, which was a minor deviation from the originally proposed research that resulted from new sequence data. A silencing construct also was completed using a promoter known to express in female reproductive tissues of cereals. Multiple microprojectile bombardment experiments to introduce these DNAs have been initiated. PARTICIPANTS: Peggy Ozias-Akins, Professor and PI, and Joann A. Conner, Assistant Research Scientist and co-PI are jointly directing the project. Dr. Conner is designing and constructing plasmid vectors. Dr. Mookkan Muruganantham, postdoc, is conducting tissue culture, microprojectile bombardment, plant regeneration, selection, and learning plasmid construction techniques. Dr. Dirk Becker, University of Hamburg, Germany, has collaborated by providing a promoter component for the silencing construct. All UGA participants in the project have taken advantage of professional development activities offered through the Plant Center, University of Georgia. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Tissue cultures capable of plant regeneration have been routinely obtained from immature embryos of tetraploid pearl millet. The outcome of selection for transgenic lines is not known at the time of this report. No impact yet to report.
Publications
- No publications reported this period
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