Progress 10/01/12 to 09/30/17
Outputs
Target Audience:This project has a diverse audience of public and private groups. The research on interspecific incompatibility and mating preference in plants is of interest to geneticists and plant breeders as well as cell and plant reproductive biologists. The research on producing seedless varieties of popular but invasive landscape plants impacts private industry, specifically the green industry including ornamental, nursery and landscape plant producers, landscape designers and installers and consumers. Efforts to provide information include formal classroom teaching in undergraduate and graduate courses. The most significant learning opportunities include an upper-level undergraduate course (Applications of Biotechnology to Plant Improvement) and an introductory course in Plant Propagation. Other efforts to reach target audiences include popular press publications and presentations. Changes/Problems:Plant material treated with high-energy gamma or fast neutron irradiation takes many years to flower. Flowering is the time the plants can be assessed for seedlessness. This extends the time required to develop seedless varieties in perennial plants. In addition, the assessment of the original plants continues for several years to ensure seedlessness over many years. Time is also required to assess vegetatively propagated individuals (rooted cuttings) from the original selections to determine whether they also are seedless. Thus, these experiments are long term. What opportunities for training and professional development has the project provided?A goal of this project is to train undergraduate and graduate students, research fellows and post-doctoral scholars. The Principal Investigator supervised all research and publication production as well as developing support and complementary funding for the project. Undergraduates work with more senior lab personal in the design, execution and analysis of experiments. The undergraduates are active in several majors at the University of Minnesota. Experimentation is excellent training in the scientific process, communication, writing and organization. The undergraduates researched in several areas closely aligned with this project including measuring the mating preferences of Nicotiana tabacum by measuring pollen tube growth, assessing genetic structure of non-native invasive plants, propagating plants using in vitro tissue culture and transforming plants. A graduate student was directly involved with research on prezygotic interspecific barriers and speciation in Nicotiana. This research provides training in the areas of gene expression, genetic modification and cell biology. A post-doctoral research fellow was key in the research to develop seedless varieties of invasive, but popular landscape plants and regulation of pollen tube growth. Professional development was provided for the postdoctoral Research Associates and graduate studentsin diversity and bioinformatics training as well as attending nation professional meetings. How have the results been disseminated to communities of interest?Results from this project were disseminated directly through refereed publications, local and national presentations, posters at professional society meetings, discussions with clientele groups, and educational outlets through classroom teaching and undergraduate and graduate student involvement in research. Results were also indirectly disseminated through research grant proposals for continued and increased funding. These include research proposals to federal agencies, for example NSF and non-governmental agencies such as foundations and industry. The applied research of the project in the production of noninvasive plant varieties is especially well suited for dissemination to a broad group of interested public, which also provides an educational opportunity. Other means of dissemination include seminars, poster presentations and meeting with colleagues and collaborators at the University of Minnesota. The formal and informal reporting of research results builds a foundation for others to follow our progress and evaluate successes. Two undergraduates were involved in a significant research experience, which is an opportunity for research dissemination. More formal education that included classroom and laboratory exercises were also form of dissemination. What do you plan to do during the next reporting period to accomplish the goals?Research to understand the fundamental mechanisms regulating pollen tube growth will use a novel approach to study the conribution of the stigma and the transmitting tract (the pollen tube growth pathway through the style) to the regulation of intraspecific pollen tube growth. We will utilize the in vivo and in vitro pollen tube growth assays developed for Nicotiana by this reserch project.. Plant material treated with high-energy gamma or fast neutron irradiation will be moved from the greenhouse to the field for continued growth, maturation and evaluation of fertility. Mutagenized material is flowering and seed set is being evaluated. Training of undergraduate and graduate students, technical staff and postdocs will continue to accomplish educational fand research objectives. Research results will be widely distributed to academic, industry and public audiences and extramural funding will be sought for associated research projects that would be synergistic to this project.
Impacts
What was accomplished under these goals?
Pollen production and delivery to the flower initiates sexual reproduction in plants. During this process, the DNA of the paternal parent (pollen) is combined with that of the female parent (ovule within the pistil). This process is essential for the production of seeds and fruit and therefore, a clear understanding of the mechanisms and biology is important for ensuring food security. In addition, sexual reproduction in plants is a method to introduce new traits into cultivated plants increasing the diversity and tolerance of our crops to changing conditions and the attack by pathogens. Many times, the traits for improvement of a crop may not reside in the same species. In this situation, an interspecific cross must be made to introduce a trait from one species into another. However, crosses between species may not provide seed for continued selection due the presence of interspecific reproductive barriers that prevent pollen tube growth. This research has investigated the mechanisms that can result in interspecific barriers and discovered the mechanism and genes that produce a reproductive barrier. We developed Nicotiana tabacum as a model to study sexual reproduction and reproductive barriers because of the many species within the genus that have diverse reproductive interactions. A unique semi-in vivo pollen tube growth assays was developed to study these interactions. Using this model system led to the discovery of a novel mechanism that is essential for interspecific incompatibility. Knowing the mechanism allowed a biotechnology approach to knockout the system, which allowed seed set between two formerly incompatible species. These results have value to basic biology in understanding reproductive barriers and the evolution of species and have application to plant improvement by enabling wide crosses and adding variation from which selection of superior types can be made. Toward the goal of discovering mechanisms of interspecific incompatibility in Nicotiana it was determined that the class III pistil-specific extensin-like protein controlled pollen tube growth. Interspecific incompatibility effectively prevents hybridization and maintains species identity. The interspecific incompatibility activity of the pistil slowed and then arrested pollen tube growth. The class III pistil-specific extensin-like protein was discovered to be the inhibitor of interspecific pollen tubes. To discover other mechanisms that regulate plant reproduction and specifically pollen tube growth, an intraspecific pollination diallel was performed. Genotypes were selected to represent the diversity of the N. tabacum germplasm collection. An in vivo pollen tube growth assay developed in our previous research was performed. Pollen tubes were measured and mean pollen tube growth calculated. These data showed there is great diversity in pollen tube growth within a species and that both the paternal and maternal genomes contribute to the regulation of the pollen tube's growth rate. This research continues to use intraspecific genetic diversity in N. tabacum pollen-pistil interactions to identify genes and alleles that facilitate or retard pollen tube growth. Ultimately, this research could provide the genetic mechanisms for parental crossing behaviors that plant breeders categorize into general or specific combining ability. The pistil of N. tabacum regulates pollen tube growth in part by secreting the pistil extensin-like protein III, the transmitting-tract-specific (TTS) protein and the 120 kDa protein (120 K) into the stylar extracellular matrix. These arabinogalactan proteins are important in regulating the growth of pollen tubes and therefore whether fertilization and seed set occurs. The N-terminal domain (NTD) of the stylar AGPs is proline rich and polymorphic among Nicotiana spp. The C-terminal domain (CTD) contains an Ole e 1-like domain, predicted to form beta-sheets that are similar in position and length among Nicotiana species. Through this research, it was determined that the TTS protein had the greatest amino acid and O-glycosylation conservation and that the class III pistil-specific extensin-like protein has the least. The dn/ds ratio for individual Nicotiana species ranged from 0.4 to 0.9 and from 0.1 to 0.8, for the class III pistil-specific extensin-like protein and TTS genes, respectively. These data indicate that class III pistil-specific extensin-like protein and TTS genes may be constrained during evolution because of their important functions in reproduction. During this analysis, a novel arabinogalactan encoding gene was discovered in Nicotiana tabacum. The gene was named Proline Rich Protein (NtPRP) and was found to have similar intron-exon configuration and protein structure to that of the other stylar arabinogalactan genes, particularly TTS. Because of its importance to plant reproduction in regulating pollen tube growth, the class III pistil-specific extensin-like protein was investigated to determine how its NTD and CTD contribute to regulation of pollen tube growth. In plants where the class III pistil-specific extensin-like protein had been eliminated, four different chimeric arabinogalactan proteins were introduced to test whether they could compliment the lost class III pistil-specific extensin-like protein function. The NTD of PIII with either the PIII or TTS CTD complemented the loss of normal class III pistil-specific extensin-like protein. However, the TTS NTD with the PIII CTD did not complement the loss of the class III pistil-specific extensin-like protein. These data support the conclusion that the the class III pistil-specific extensin-like protein NTD is necessary for regulation of pollen tube growth and fertilization. A goal of this research is to mitigate the impact that non-native invasive plants on the environment. The approach is to use mutagenesis breeding to produce seedless varieties of popular, but invasive plants. Seeds and unrooted cuttings were selected to undergo irradiation treatments. However, no published data on radiation sensitivity of seeds or unrooted cuttings from Acer ginnala or Berberis thunbergii were available. Therefore, a wide range of irradiation was used. Treated material was grown in Cottage Grove, MN. Acer ginnala was evaluated in late spring for flowering and fruit production in plant populations that were 8-10 years old. Although the level of seed set varied among mutagenized individuals, the vast majority had a high level of seed set. Six Acer ginnala individuals were noted as having not flowered or set seed over a three-year period, while others in the population were flowering and setting seed. Further research on the A.cer ginnala selections showed they could be efficiently propagated by rooting green shoots. Two seedless Berberis thunbergii were selected and several individual with greatly reduced viable seed production were noted. This research has developed new seedless germplasm that can be used to replace the non-native and invasive Acer ginnala and Berberis thunbergii, currently being sold. The research described above has resulted in the continued training and education of one post-doctoral technician, one graduate student and two undergraduate students.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Noyszewski, A.K., Y-. Liu, C.M.L. Alves, and A.G. Smith. 2017. Polymorphism and structure of stylespecific arabinogalactan proteins of Nicotiana as determinants of pollen tube growth. Submitted to BMC Genomics, 17:186, https://doi.org/10.1186/s12862-017-1011-2.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Smith, A.G. 2017. MNLA Foundation Grant Update - Development of Seedless Varieties of Popular, but Invasive Plants. 2017 Northern Green.
- Type:
Other
Status:
Published
Year Published:
2017
Citation:
Noyszewski, A.K., A.G. Smith, N.O. Anderson. 2017. Native/Exotic Reed Canarygrass Management and Native American Treaty Rights. University of Minnesota GenoFest 2017
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Alves, C.M.L., A.K. Noyszewski and A.G. Smith. 2017. Pollen tube growth regulation and crop improvement, DuPont Plant Science Symposium Plant Breeding: Rooted in Education, St Paul, MN.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2017
Citation:
Smith, A.G. and A.K. Noyszewski. 2017. Mutagenesis Breeding for Seedless Varieties of Popular Landscape Plants. Acta Horticulturae, in press.
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:This project has a diverse audience of public and private groups. The research on interspecific incompatibility is of interest to geneticists and plant breeders as well as cell and plant reproductive biologists. The research on producing seedless varieties of popular but invasive landscape plants impacts private industry, specifically the green industry including ornamental, nursery and landscape plant producers, landscape designers and installers and consumers. Efforts to provide information include formal classroom teaching in undergraduate and graduate courses. The most significant learning opportunities include an upper-level undergraduate course (Applications of Biotechnology to Plant Improvement) and an introductory course in Plant Propagation. Other efforts to reach target audiences include popular press publications and presentations. Changes/Problems:Research to understand the fundamental mechanisms of interspecific compatibility and regulation of pollen tube growth will use a novel approach to alter arabinogalactan structure to measure the effect of these changes on regulation of pollen tube growth. These will include both in vivo and in vitro experimentation. Plant material treated with high-energy gamma or fast neutron irradiation will be moved from the greenhouse to the field for continued growth, maturation and evaluation of fertility. Mutagenized material is flowering and seed set is being evaluated. Training of undergraduate and graduate students and postdocs will continue to accomplish educational objectives and accomplish research objectives. Research results will be widely distributed to academic, industry and public audiences and extramural funding will be sought for associated research projects that would be synergistic to this project. What opportunities for training and professional development has the project provided?A goal of this project is to train undergraduate and graduate students, research fellows and post-doctoral scholars. The Principal Investigator supervised all research and publication production as well as developed support and complementary funding for the project. Undergraduates work with more senior lab personal in the design, execution and analysis of experiments. The undergraduates are active in several majors. Experimentation is excellent training in the scientific process, communication, writing and organization. The undergraduates researched in several areas closely aligned with this project including measuring the mating preferences Nicotiana tabacum by monitoring pollen tube growth in reciprocal crosses, developing an undergraduate laboratory experience in testing for GMO DNA in food products that will be sued to develop a teaching lab exercise, measurement of Nicotiana pollen tubes grown in vitro and in vitro propagation and transformation of Nicotiana species other than N. tabacum. A graduate student was directly involved with research on prezygotic interspecific barriers and speciation in Nicotiana. This research provides training in the area of gene expression, genetic modification and cell biology. Professional development was provided for a M.S. level technician and a postdoctoral Research Associate in the department of Horticultural Science. The M.S. level technician was trained in areas of mutagenesis breeding for seedless plant production and reproductive biology of plants, 3' and 5' RACE for nucleotide sequence of cDNA clones for three arabinogalactan genes as well as genomic nucleotide sequencing for several Nicotiana species. These data contribute directly to the understanding of the regulation of pollen tube growth. A postdoctoral Research Associate was trained in the design, production and testing of several plant gene constructs for the manipulation of pollen tube growth in specific females engineered for pollen interactions. These positions are key to advancing the project in the areas of noninvasive plant production through selection of noninvasive varieties and the understanding the pollen-pistil interactions controlling interspecific incompatibility and pollen tube growth. The research in the production of noninvasive varieties has important partners in the horticultural and green industries that provide broader training opportunities. These organizations have common interests and goals that provide direct and in-kind funding and collaboration to accomplish research support. Therefore the collaborators are informed of the project progress and strategies for continued progress are time for education. How have the results been disseminated to communities of interest?Results from this project were disseminated directly through refereed publications, local and national presentations, posters at professional society meetings, discussions with clientele groups, and educational outlets through classroom teaching and undergraduate and graduate student involvement in research. Results were also indirectly disseminated through research grant proposals for continued and increased funding. These include research proposals to federal agencies, for example NSF and non-governmental agencies such as foundations and industry. The applied research of the project in the production of noninvasive plant varieties is especially well suited for dissemination to a broad group of interested public, which also provides an educational opportunity. Other means of dissemination include seminars, poster presentations and meeting with colleagues and collaborators at the University of Minnesota. The formal and informal reporting of research results builds a foundation for others to follow our progress and evaluate successes. Three undergraduates were involved in a significant research experience, which is an opportunity for research dissemination. More formal education that included classroom and laboratory exercises were also a form of dissemination. What do you plan to do during the next reporting period to accomplish the goals?Research to understand the fundamental mechanisms of interspecific compatibility and regulation of pollen tube growth will use a novel approach to alter arabinogalactan structure to measure the effect of these changes on regulation of pollen tube growth. These will include both in vivo and in vitro experimentation. Plant material treated with high-energy gamma or fast neutron irradiation will be moved from the greenhouse to the field for continued growth, maturation and evaluation of fertility. Mutagenized material is flowering and seed set is being evaluated. Training of undergraduate and graduate students and postdocs will continue to accomplish educational objectives and accomplish research objectives. Research results will be widely distributed to academic, industry and public audiences and extramural funding will be sought for associated research projects that would be synergistic to this project.
Impacts
What was accomplished under these goals?
This project contains four separate, but interconnected objectives. The objectives are: 1. Discovering mechanisms of interspecific incompatibility in Nicotiana. Geographic, ecological and genetic processes isolate plant populations resulting in the evolution and maintenance of species. In plants, interspecific incompatibility between pollen and pistil is a prezygotic reproductive barrier that regulates fertilization and contributes to the evolution and maintenance of species. Previously, this project identified the Class III Pistil Extension-Like protein as a regulator of interspecific incompatibility among Nicotiana species. Identification of genes that regulate interspecific incompatibility will provide new insights into how species evolve, how species are maintained, mechanisms that regulate hybridization and factors that regulate gene flow. Several transgenic lines with reduced arabinogalactan protein accumulation or modified arabinogalactan protein accumulation were produced. These lines are being analyzed to correlate the modified arabinogalactan with changes in pollen tube growth. This objective has direct application to horticulture and using genetically diverse germplasm via interspecific crosses for plant improvement. Understanding the mechanisms that regulate speciation will enhance our understanding of crop plant evolution and how to manipulate reproduction for introgression of genes among taxa. 2. Transcriptome analysis of pollen tubes growing in compatible versus incompatible styles and discovery of genes producing the transmitting tissue extracellular matrix. This objective will identify genes important to pollen tube growth and interspecific reproductive barriers. Pollen tubes are a unique cell type of the plant and are known to accumulate unique transcripts and proteins associated with unique function. However, little is known about the differential expression that takes place in the pollen tubes during compatible versus incompatible pollination. Our evidence suggests that the transmitting tissue (TT) of the pistil is key to regulating many aspects of pollen tube growth. We hypothesize that identification of transcripts specific to the TT will lead to the identification of secreted proteins that contribute to the extracellular matrix of the TT and function in the regulation of pollen tube growth. We have collected over 17 genotypes (some current or former cultivars) of N. tabacum that are representative of the germplasm diversity in the U.S.A collection. This diverse collection is being characterized for mating preferences by measuring pollen tube growth among the female genotypes. Identification of compatible and incompatible genotypes is a first step toward discovering genes that regulate pollen tube growth in the male gamete and female TT. Application of this information is important to facilitating pollination and therefore seed and fruit production in diverse genotypes and environments as well as limiting fertilization from undesirable pollinators. 3. Developing strategies to eliminate invasiveness of popular landscape plants. Invasive species are a primary threat to biodiversity on the planet, second only to habitat destruction, and are one of the least reversible of all human impacts on the environment. They threaten natural areas by competing with native taxa, altering habitats, introducing new genes or alleles to populations, and disturbing ecosystem processes. Such impacts frequently result in the displacement and eventual elimination of native plants and animals, along with their unique genetic resources and ecological functions. It is extremely difficult to remove invasive species once they become established. This objective will develop strategies to eliminate the potential for invasiveness from popular landscape plants. Several individuals of Acer ginnala and Berberis thunbergii were identified as producing no or significantly less seed after mutagenesis. These plants are being propagated for testing in several environments. 4. Train undergraduate and graduate students, research fellows and post-doctoral scholars.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
McGinnis, E.E., A.G. Smith, M.H. Meyer. Environmental Control of Flowering in Pennsylvania Sedge. Submitted to Botany.
Smith, A.G. and A.K. Noyszewski. 2016. =Mutagenesis Breeding for Seedless Varieties of Popular Landscape Plants. Submitted to Acta Horticulturae.
Noyszewski, A.K., Y-. Liu, C.M.L. Alves, and A.G. Smith. 2016. Polymorphism and strucure of stylespecific arabinogalactan proteins of Nicotiana as determinants of pollen tube growth. Submitted to BMC Genomics.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Smith, A.G. 2016. University of Wisconsin, River Falls, ANSC222 Introduction to Biotechnology, seminar on Plant Biotechnology.
Smith, A.G.. 2016. The Third International Symposium on Woody Ornamentals for the Temperate Zone. Mutagenesis Breeding for Seedless Varieties of Popular Landscape Plants.
Noyszewski, A.K., Y-. Liu, C.M.L. Alves, and A.G. Smith. 2016. Sequence characterization of stylar arabinogalactan genes from distantly related Nicotiana species as determinants of pollen tube growth. Research Collaboration Network Meeting, Phoenix AZ.
Alves, C.M.L., A.K. Noyszewski, Y-c. Liu, and A.G. Smith. 2016. Regulation of Pollen Tube Growth in Nicotiana. Annual Meeting of the American Society of Plant Biologists.
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:This project has a diverse audience of public and private groups. The research on interspecific incompatibility is of interest to geneticists and plant breeders as well as cell and plant reproductive biologists. The research on eliminating invasiveness of popular landscape plants impacts private industry, specifically the green industry including ornamental, nursery and landscape plant producers, landscape designers and installers and consumers. Efforts to provide information include formal classroom teaching in undergraduate and graduate courses. The most significant learning opportunities include an upper-level undergraduate course (Applications of Biotechnology to Plant Improvement), an introductory course in Plant Propagation. Other efforts to reach target audiences include popular press publications and presentations. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A goal of this project is to train undergraduate and graduate students, research fellows and post-doctoral scholars. The Principal Investigator supervised all research and publication production as well as developed support and complementary funding for the project. Undergraduates work with more senior lab personal in the design, execution and analysis of experiments. The undergraduates are active in several majors. Experimentation is excellent training in the scientific process, communication, writing and organization. The undergraduates researched in several areas closely aligned with this project including measuring the mating preferences Nicotiana tabacum by monitoring pollen tube growth in reciprocal crosses, developing an undergraduate laboratory experience in testing for GMO DNA in food products that will be sued to develop a teaching lab exercise, measurement of Nicotiana pollen tubes grown in vitro and in vitro propagation and transformation of Nicotiana species other than N. tabacum. A graduate student was directly involved with research on prezygotic interspecific barriers and speciation in Nicotiana. This research provides training in the area of gene expression, genetic modification and cell biology. Professional development was provided for a M.S. level technician and a postdoctoral Research Associate in the department of Horticultural Science. The M.S. level technician was trained in areas of mutagenesis breeding for seedless plant production and reproductive biology of plants, 3' and 5' RACE for nucleotide sequence of cDNA clones for three arabinogalactan genes as well as genomic nucleotide sequencing for several Nicotiana species. These data contribute directly to the understanding of the regulation of pollen tube growth. A postdoctoral Research Associate was trained in the design, production and testing of several plant gene constructs for the manipulation of pollen tube growth in specific females engineered for pollen interactions. These positions are key to advancing the project in the areas of noninvasive plant production through selection of noninvasive varieties and the understanding the pollen-pistil interactions controlling interspecific incompatibility and pollen tube growth. The research in the production of noninvasive varieties has important partners in the horticultural and green industries that provide broader training opportunities. These organizations have common interests and goals that provide direct and in-kind funding and collaboration to accomplish research support. Therefore the collaborators are informed of the project progress and strategies for continued progress are time for education. How have the results been disseminated to communities of interest?Results from this project were disseminated directly through refereed publications, local and national presentations, posters at professional society meetings, discussions with clientele groups, and educational outlets through classroom teaching and undergraduate and graduate student involvement in research. Results were also indirectly disseminated through research grant proposals for continued and increased funding. These include research proposals to federal agencies, for example NSF and non-governmental agencies such as foundations and industry. The applied research of the project in the production of noninvasive plant varieties is especially well suited for dissemination to a broad group of interested public, which also provides an educational opportunity. Other means of dissemination include seminars, poster presentations and meeting with colleagues and collaborators at the University of Minnesota. The formal and informal reporting of research results builds a foundation for others to follow our progress and evaluate successes. Three undergraduates were involved in a significant research experience, which is an opportunity for research dissemination. More formal education that included classroom and laboratory exercises were also a form of dissemination. What do you plan to do during the next reporting period to accomplish the goals?Research to understand the fundamental mechanisms of interspecific compatibility and regulation of pollen tube growth will use a novel approach to alter arabinogalactan structure to measure the effect of these changes on regulation of pollen tube growth. These will include both in vivo and in vitro experimentation. Plant material treated with high-energy gamma or fast neutron irradiation will be moved from the greenhouse to the field for continued growth, maturation and evaluation of fertility. Mutagenized material is flowering and seed set is being evaluated. Training of undergraduate and graduate students and postdocs will continue to accomplish educational objectives and accomplish research objectives. Research results will be widely distributed to academic, industry and public audiences and extramural funding will be sought for associated research projects that would be synergistic to this project.
Impacts
What was accomplished under these goals?
Listed below are the goals of the project as outlined in the Project Initiation section. This project contains four separate, but interconnected objectives. The objectives are: 1. Discovering mechanisms of interspecific incompatibility in Nicotiana. Geographic, ecological and genetic processes isolate plant populations resulting in the evolution and maintenance of species. In plants, interspecific incompatibility between pollen and pistil is a prezygotic reproductive barrier that regulates fertilization and contributes to the evolution and maintenance of species. Previously, this project identified the Class III Pistil Extension-Like protein as a regulator of interspecific incompatibility among Nicotiana species. Identification of genes that regulate interspecific incompatibility will provide new insights into how species evolve, how species are maintained, mechanisms that regulate hybridization and factors that regulate gene flow. Several transgenic lines with reduced arabinogalactan protein accumulation or modified arabinogalactan protein accumulation were produced. These lines are being analyzed to correlate the modified arabinogalactan with changes in pollen tube growth. This objective has direct application to horticulture and using genetically diverse germplasm via interspecific crosses for plant improvement. Understanding the mechanisms that regulate speciation will enhance our understanding of crop plant evolution and how to manipulate reproduction for introgression of genes among taxa. 2. Transcriptome analysis of pollen tubes growing in compatible versus incompatible styles and discovery of genes producing the transmitting tissue extracellular matrix. This objective will identify genes important to pollen tube growth and interspecific reproductive barriers. Pollen tubes are a unique cell type of the plant and are known to accumulate unique transcripts and proteins associated with unique function. However, little is known about the differential expression that takes place in the pollen tubes during compatible versus incompatible pollination. Our evidence suggests that the transmitting tissue (TT) of the pistil is key to regulating many aspects of pollen tube growth. We hypothesize that identification of transcripts specific to the TT will lead to the identification of secreted proteins that contribute to the extracellular matrix of the TT and function in the regulation of pollen tube growth. We have collected over 17 genotypes (some current or former cultivars) of N. tabacum that are representative of the germplasm diversity in the U.S.A collection. This diverse collection is being characterized for mating preferences by measuring pollen tube growth among the female genotypes. Identification of compatible and incompatible genotypes is a first step toward discovering genes that regulate pollen tube growth in the male gamete and female TT. Application of this information is important to facilitating pollination and therefore seed and fruit production in diverse genotypes and environments as well as limiting fertilization from undesirable pollinators. 3. Developing strategies to eliminate invasiveness of popular landscape plants. Invasive species are a primary threat to biodiversity on the planet, second only to habitat destruction, and are one of the least reversible of all human impacts on the environment. They threaten natural areas by competing with native taxa, altering habitats, introducing new genes or alleles to populations, and disturbing ecosystem processes. Such impacts frequently result in the displacement and eventual elimination of native plants and animals, along with their unique genetic resources and ecological functions. It is extremely difficult to remove invasive species once they become established. This objective will develop strategies to eliminate the potential for invasiveness from popular landscape plants. Several individuals of Acer ginnala and Berberis thunbergii were identified as producing no or significantly less seed after mutagenesis. These plants are being propagated for testing in several environments. 4. Train undergraduate and graduate students, research fellows and post-doctoral scholars.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Li1, J., S. Dukowic-Schulze1 I.E. Lindquist, A.D. Farmer, B. Kelly, T. Li, Al.G. Smith, E.F. Retzel, J Mudge and C. Chen. 2015. The plant-specific protein FEHLSTART controls male meiotic entry, initializing meiotic synchronization in Arabidopsis. Plant J, DOI: 10.1111/tpj.13026.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
McGinnis, E.E., A.G. Smith, M.H. Meyer. Environmental Control of Flowering in Pennsylvania Sedge. Submitted to Botany.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: This project has a diverse audience of public and private groups. The research on interspecific incompatibility is of interest to geneticists and plant breeders as well as cell and plant reproductive biologists. The research on eliminating invasiveness of popular landscape plants impacts private industry, specifically the green industry including ornamental, nursery and landscape plant producers, landscape designers and installers and consumers. Efforts to provide information include formal classroom teaching in undergraduate and graduate courses. The most significant learning opportunities include an upper-level undergraduate course (Applications of Biotechnology to Plant Improvement), an introductory course in Plant Propagation and a graduate level courses (Manipulation of Plant Growth and Reproduction, Research Ethics in the Plant and Environmental Sciences). Other efforts to reach target audiences include popular press publications and presentations. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A goal of this project is to train undergraduate and graduate students, research fellows and post-doctoral scholars. The Principal Investigator supervised all research and publication production as well as developed support and complementary funding for the project. Undergraduates work with more senior lab personal in the design, execution and analysis of experiments. The undergraduates are active in several majors. Experimentation is excellent training in the scientific process, communication, writing and organization. The undergraduates researched in several areas closely aligned with this project including assessment of pollen tube growth in vitro that tested the uptake and regulation of growth by specific proteins and gene introduction. Researched focused on the cultural and environmental effects to optimize gene introduction in Nicotiana spp., which is very important to the understanding of interspecific reproductive barriers. An undergraduate student was very important in the first assessment of fast neutron mutagenesis for the production of seedless varieties for the production of invasive plants and anther student is researching the detection of genetically modified plant products in food. A graduate student was directly involved with research on prezygotic interspecific barriers and speciation in Nicotiana. This research provides training in the area of gene expression, genetic modification and cell biology. A postdoctoral Research Associate in the department of Horticultural Science was trained in the areas of mutagenesis breeding for seedless plant production and reproductive biology of plants. This position is key to advancing the project in the areas of noninvasive plant production through selection of noninvasive varieties and the understanding the pollen-pistil interactions controlling interspecific incompatibility and pollen tube growth. The research associate is applying research derived information to manage gene flow among plants. The research in the production of noninvasive varieties has important partners in the horticultural and green industries that provide broader training opportunities. These organizations have common interests and goals that provide direct and in-kind funding and collaboration to accomplish research support. Therefore the collaborators are informed of the project progress and strategies for continued progress are time for education. How have the results been disseminated to communities of interest? Results from this project were disseminated directly through refereed publications, local and national presentations, posters at professional society meetings, discussions with clientele groups, and educational outlets through classroom teaching and undergraduate and graduate student involvement in research. Results were also indirectly disseminated through research grant proposals for continued and increased funding. These include research proposals to federal agencies, for example NSF and non-governmental agencies such as foundations and industry. The applied research of the project in the production of noninvasive plant varieties is especially well suited for dissemination to a broad group of interested public, which also provides an educational opportunity. Other means of dissemination include seminars, poster presentations and meeting with colleagues and collaborators at the University of Minnesota. The formal and informal reporting of research results builds a foundation for others to follow our progress and evaluate successes. A publication in the Minnesota Nursery and Landscape Association’s publication the “SCOOP” described our strategy and progress in the production of seedless verities of invasive landscape plants. Three undergraduates were involved in a significant research experience, which is an opportunity for research dissemination. More formal education that included classroom and laboratory exercises were also a form of dissemination. What do you plan to do during the next reporting period to accomplish the goals? Research to understand the fundamental mechanisms of interspecific compatibility and regulation of pollen tube growth will use a novel approach to alter arabinogalactan structure to measure the effect of these changes on regulation of pollen tube growth. These will include both in vivo and in vitro experimentation. Plant material treated with high-energy gamma or fast neutron irradiation will be moved from the greenhouse to the field for continued growth, maturation and evaluation of fertility. Mutagenized material is flowering and seed set is being evaluated. Training of undergraduate and graduate students and postdocs will continue to accomplish educational objectives and accomplish research objectives. Research results will be widely distributed to academic, industry and public audiences and extramural funding will be sought for associated research projects that would be synergistic to this project.
Impacts
What was accomplished under these goals?
Plant growth and reproduction are essential to the development of seeds and fruits, which are critical processes for horticulture, plant productivity and directly contributes to plant improvement and diversity of crops. However, plant reproduction can lead to some plant species escaping cultivation and becoming invasive. Invasive species are a primary threat to biodiversity on the planet, second only to habitat destruction, and are one of the least reversible of all human impacts on the environment. They threaten natural areas by competing with native taxa, altering habitats, introducing new genes or alleles to populations, and disturbing ecosystem processes. Invasive plants cause both economic and environmental damage. Researching genes important to growth, reproduction and the evolution of species provides a basis to manipulate these processes for crop improvement and to produce noninvasive varieties of popular, but invasive plants. 1. Discovering mechanisms of interspecific incompatibility in Nicotiana. Interactions between the pollen (male gametes of plants) and pistil (female reproductive organ of plants) determine pollination compatibilities that function to allow hybrids to form or maintain separate species by acting as reproductive barriers. Pollen tube growth and fertilization and seed set is central to yield, essential to the ornamental value of landscape plant, a major determinant of a plant's invasiveness, and is the route for gene flow from genetically modified plants to weedy species or unintended crops. This research project has increased the understanding of stamen and pistil development, the function of genes in reproduction, and mechanisms that control organ-specific expression. These data can be used in applications to facilitate wide crosses between species for plant breeding and improvement and to produce plants with reduced invasiveness. This research will also produce information to better understand the nature of invasive plants as well as develop strategies to manage gene flow. Toward the goal of discovering mechanisms of interspecific reproductive barriers, a small family of arabinogalactan proteins was identified as key regulators of pollen tube growth and fertilization. The mechanisms by which the arabinogalactan proteins regulate pollen tube growth and therefore fertilization were investigated. The arabinoglactan genes play an important role in the evolution of the genus Nicotiana because they regulate reproductive compatibility among species. We have identified conserved and divergent regions of these proteins and are testing these regions for functions in reproduction. These data are important in understanding how wide crosses can be facilitated to introduce novel genes into plants as well as how speciation occurs and is maintained. 2. Transcriptome analysis of pollen tubes growing in compatible versus incompatible styles and discovery of genes producing the transmitting tissue extracellular matrix. This objective will identify genes important to pollen tube growth and interspecific reproductive barriers. We hypothesize that there are many gene are involved in the regulation of pollen tube growth. Some of the pollen tube regulatory products interact directly with the arabinogalactan proteins and others act independently. We are using RNA-seq analysis to identify RNAs that are differentially accumulated between pollen tubes growth in a compatible vs. an incompatible pistil. The identified RNAs will be used to understand how pollen tube growth can be facilitated or inhibited, depending on the interaction between the pollen and pistil genotypes. 3. Developing strategies to eliminate invasiveness of popular landscape plants. Invasive species are a primary threat to biodiversity on the planet, second only to habitat destruction, and are one of the least reversible of all human impacts on the environment. Developing seedless cultivars of landscape plants would benefit all segments of the nursery and landscape industry. This research is focused on producing seedless varieties of Berberis thunbergii (Japanese barberry), Acer ginnala (Amur maple), Acer platanoides (Norway maple) and Euonymus alatus (Winged euonymus). These are popular landscape plants that were intentionally introduced, but are known to be invasive. Our approach is using high-energy gamma or fast neutron irradiation treatments of seeds and unrooted cuttings to create mutation and chromosomal damage that results in seedlessness. Seedlessness and novel phenotypes are selected from seeds and cuttings that have undergone radiation treatments that result in 50% lethality. This level of mutagenesis creates maximum variation for seedless, dwarf and novel plant selection while allowing sufficient numbers of plants to survival for trialing. All plants were evaluated for flowering and seed set. Plants exhibiting novelty (unique growth form, leaf shape, leaf color, flower color, etc.) were documented and these plants will be monitored closely.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
McGinnis, E.E., A.G. Smith, and M.H. Meyer. 2014. A post-floral initiation chilling treatment controls dichogamy sequence in Carex pensylvanica. Hort Tech 24:301-306.
- Type:
Other
Status:
Published
Year Published:
2014
Citation:
Noyszewski, A.K. and A.G. Smith. 2014. Breeding for noninvasive varieties. SCOOP. 37: 27-32.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: This project has a diverse audience of public and private groups. The research on interspecific incompatibility is of interest to geneticists and plant breeders as well as cell and plant reproductive biologists. The research on eliminating invasiveness of popular landscape plants impacts private industry, specifically the green industry including ornamental, nursery and landscape plant producers, landscape designers and installers and consumers. Efforts to provide information include formal classroom teaching in undergraduate and graduate course offerings. The most significant learning opportunities include an upper-level undergraduate course (Applications of Biotechnology to Plant Improvement) and a graduate level courses (Manipulation of Plant Growth and Reproduction, Research Ethics in the Plant and Environmental Sciences). Other efforts to reach target audiences include publications and presentations. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A goal of this project is to train undergraduate and graduate students, research fellows and post-doctoral scholars. The Principal Investigator supervised all research and publication production as well as developed support and complementary funding for the project. Undergraduates work with more senior lab personal in the design, execution and analysis of experiments. The undergraduates are active in several majors including Horticulture, Applied Plant Sciences, Cell and Development and Biology and Society and Environment. Experimentation is excellent training in the scientific process, communication, writing and organization. The undergraduates worked in several areas aligned with this project including assessment of pollen tube growth in vitro that tested the uptake and regulation of growth by specific proteins and in vivo pollen tube growth during heat stress. Two students researched cultural and environmental effects to optimize gene introduction in Nicotiana spp., which is very important to the understanding of interspecific incompatibility. An undergraduate student was very important in the first assessment of fast neutron mutagenesis for the production of seedless varieties for the production of invasive plants and one student researched issues in the acceptance and labeling of genetically engineered crops. Two graduate students were directly involved with this research. One student graduated with an Applied Plant Sciences Ph.D. in 2013 writing a thesis focused on environmental control of flowering and legal issues in the regulation of GMO crops. A new student began in 2013 with research focused on researching prezygotic interspecific barriers and speciation in Nicotiana. This research provides training in the area of gene expression, genetic modification and cell biology. A postdoctoral Research Associate in the department of Horticultural Science was trained in the areas of mutagenesis breeding for seedless plant production and reproductive biology of plants. This position is key to advancing the project in the areas of noninvasive plant production through selection of noninvasive varieties and the understanding the pollen-pistil interactions controlling interspecific incompatibility and pollen tube growth. The research in the production of noninvasive varieties has important partners in the horticultural and green industries that provide broader training opportunities. These organizations have common interests and goals that provide direct and in-kind funding and collaboration to accomplish research support. Therefore the collaborators are informed of the project progress and strategies for continued progress are time for education. How have the results been disseminated to communities of interest? Results from this project were disseminated directly through refereed publications, local and national presentations, posters at professional society meetings, discussions with clientele groups, and educational outlets through classroom teaching and undergraduate and graduate student involvement in research. Results were also indirectly disseminated through research grant proposals for continued and increased funding. These include research proposals to federal agencies, for example NSF and non-governmental agencies such as foundations and industry. The applied research of the project in the production of noninvasive plant varieties is especially well suited for dissemination to a broad group of interested public, which also provides an educational opportunity. Other means of dissemination include seminars, poster presentations and meeting with colleagues and collaborators at the University of Minnesota. The formal and informal reporting of research results builds a foundation for others to follow our progress and evaluate successes. Presentations made in 2013 include: the Ornamental Plant Symposium, Chicago Botanical Gardens (Production of non-invasive ornamental plants using mutagenesis breeding); the Annual Meeting of the American Society of Plant Biologists (Nicotiana tabacum pollen nonspecifically takes up protein during hydration, germination and pollen tube growth) and at the University of Minnesota School of Public Health National Public Health Week Film Festival screening of “Genetic Roulette.” In addition to traditional classroom teaching, undergraduates are actively involved in this research. Six undergraduates were involved in a significant research experience, which is an opportunity for research dissemination. More formal education that included classroom and laboratory exercises were also a form of dissemination. What do you plan to do during the next reporting period to accomplish the goals? Research to understand the fundamental mechanisms of interspecific compatibility and regulation of pollen tube growth will use a similar approach where pollen or the pistil is modified by specific treatments or through genetic engineering. These will include both in vivo and in vitro experimentation. Plant material treated with high-energy gamma or fast neutron irradiation will be moved from the greenhouse to the field for continued growth, maturation and evaluation of fertility. Training of undergraduate and graduate students and postdocs will continue to accomplish educational objectives and accomplish research objectives. Research results will be widely distributed to academic, industry and public audiences and extramural funding will be sought for associated research projects that would be synergistic to this project.
Impacts
What was accomplished under these goals?
Plant growth and reproduction are essential to the development of seeds and fruits, which are critical processes for horticulture and plant productivity. However, plant reproduction can lead to some plant species escaping cultivation and becoming invasive. Invasive species are a primary threat to biodiversity on the planet, second only to habitat destruction, and are one of the least reversible of all human impacts on the environment. They threaten natural areas by competing with native taxa, altering habitats, introducing new genes or alleles to populations, and disturbing ecosystem processes. Invasive plants cause both economic and environmental damage. Genes important to growth, reproduction and the evolution of species were identified and analyzed to provide a basis to manipulate these processes for crop improvement and to produce noninvasive varieties of popular, but invasive plants. Interactions between the pollen (male gametes of plants) and pistil (female reproductive organ of plants) determine pollination compatibilities that function to allow hybrids to form or maintain separate species by acting as a barrier. Flower development is central to the yield many crops produce, essential to the ornamental value of landscape plant, a major determinant of a plant's invasiveness, and is the route for gene flow from genetically modified plants to weedy species or unintended crops. This research project will increase the understanding of stamen and pistil development, the function of genes in reproduction, and mechanisms that control organ-specific expression. These data are used in applications to facilitate wide crosses between species for plant breeding and improvement and to produce plants with reduced invasiveness. This research will also produce information to better understand the nature of invasive plants as well as develop strategies to eliminate the invasive potential of popular landscape plants. Toward the goal of discovering mechanisms of interspecific incompatibility, the function of the pistil in regulating pollen tube growth was tested. Genetic ablation was used to eliminate the mature transmitting tissue, a highly specialized tissue thought essential to pollen tube growth and fertilization. Despite the absence of the mature transmitting tissue and greatly reduced transmitting-tissue specific gene expression, self-pollen tubes grew to the end of the style. Pollen tubes grew at a slower rate in styles without a mature transmitting tissue. However, pollen tubes were able to grow to and fertilize ovules producing seed. Surprisingly, elimination of the transmitting tissue significantly altered interspecific pollen tube growth, implicating it as a major regulatory tissue that facilitates or inhibits interspecific pollen tube growth in a species dependent manner and in controlling prezygotic reproductive barriers. These data are important in understanding how wide crosses can be facilitated to introduce novel genes into plants as well as how speciation occurs and is maintained. The study of plant gene expression and development can lead to direct improvements in horticultural crop characters including: increased ornamental value, decreased plant invasiveness, and elimination of gene flow from genetically modified plants. This research project increases the understanding of the function of genes in reproduction and mechanisms that control organ-specific expression. Specifically, the identification of pistil-specific extensin-like protein as a regulator of interspecific incompatibility is a new concept in plant biology. It will lead to the discovery of the underlying mechanisms of reproduction functions and how plant species are evolved. Transcriptome analysis of pollen tubes growing in compatible versus incompatible styles was used to discover of genes and proteins that regulate pollen tube growth. We used Nicotiana because it is a model for research in plant reproductive biology having genetic resources, species richness, large flowers, and ease of gene introduction. To better understand pollen-pistil interactions, we used genetic ablation to produce a unique mutant lacking the highly differentiated transmitting tissue found in mature pistils. The transmitting tissue evolved with the enclosed ovules of angiosperms and is the pathway for pollen tube growth from the stigma to the ovules. It is thought to be essential for fertilization and is known to regulate the growth of pollen tubes. The genes involved in these functions are being discovered using RNA-sequencing. A significant goal for this project is to eliminate invasiveness of popular landscape plants. Developing seedless cultivars of landscape plants would benefit all segments of the nursery and landscape industry. The resultant seedless cultivars would be very popular with many consumers for several reasons. Seedless cultivars have utility in species where fruit or seed is a nuisance or unsightly, there is the potential for the plant to be invasive, the production of seed reduces growth or viability of the plant, or production of seed reduces flower number and longevity. Availability of seedless varieties of desirable landscape plant species could lead to greatly increased sales of those species. This research is focused on producing seedless varieties of Berberis thunbergii (Japanese barberry), Acer ginnala (Amur maple), Acer platanoides (Norway maple) and Euonymus alatus (Winged euonymus). These are popular landscape plants that were intentionally introduced, but are known to be invasive. Our approach is using high-energy gamma or fast neutron irradiation treatments of seeds and unrooted cuttings to create mutation and chromosomal damage that results in seedlessness. Initial research determined the relative sensitivities of a species and propagule to achieve maximum efficiency for mutagenesis. The target species are long-lived woody-perennial species that have a multi-year juvenile period, requiring selection of seedlessness over many years. Mutagenized material from 2013 is being grown in the greenhouse and will be transplanted to the field with mutagenized material from previous years irradiation treatments for assessment and selection.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Smith, A.G., A. Hummel and A.K. Noyszewski. 2013. Nicotiana tabacum pollen nonspecifically takes up protein during hydration, germination and pollen tube growth. Annual Meeting of the American Society of Plant Biologists, July, 2013.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Andrzej K. Noyszewski and Alan G. Smith. 2013. Production of non-invasvise ornamental plants using mutagenesis breeding. Ornamental Plant Symposium, Chicago Botanical Gardens. 3 October 2013.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Smith, A.G., C.A. Eberle, N.O. Anderson, B.M. Clasen, A.D. Hegeman. 2013. The transmitting tissue of Nicotiana tabacum is not essential to pollen tube growth, and its ablation can reverse prezygotic interspeci?c barriers. Plant Reprod 26:339-350.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Eberle, C.A., Neil O Anderson, Benjamin M Clasen, Alan G Smith. 2013. PELPIII the Class III Pistil-Specific Extensin-Like Nicotiana tabacum protein is essential for interspecific incompatibility. The Plant J. 74:805-814.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Outputs from this project have been disseminated directly through refereed publications, local and national presentations, posters at professional society meetings, discussions with clientele groups, and educational outlets through classroom teaching and undergraduate and graduate student involvement in research. The outputs generated by this project were also indirectly disseminated through research grant proposals for continued and increased funding. These include research proposals to federal agencies, for example NSF and non-governmental agencies such as foundations and industry. Other means of dissemination include seminars, poster presentations and meeting with colleagues and collaborators at the University of Minnesota. The formal and informal reporting of research results builds a foundation for others to follow our progress and evaluate successes. Presentations made in 2012 include: Northern Green Expo Schedule, Ask the Experts, Horticultural Crop Improvement and Reducing Invasiveness, Jan., 2012; The Pistil-specific extensin-like protein (PELP) is required for interspecific incompatibility in Nicotiana. Annual Meeting of the American Society of Plant Biologists, July 20, 2012. Ph.D. final defense seminar "Interspecific Prezygotic Reproductive Barriers in Nicotiana" and Complex Carbohydrate Research Center Course, Separation and Characterization of Glycoprotein and Glycolipid Oligosaccharides. In addition to traditional classroom teaching, undergraduates are actively involved in this research. Three undergraduates pursued independent research as well as provided valuable laboratory support. The focus of the three projects was "Determination of radiation sensitivity of invasive species for the production of sterile varieties"; "Developing Gene Introduction Protocols" and "Surveying Genes Regulating Interspecific Incompatibility". "The development and delivery of educational material and seminar presentations represent the interdisciplinary nature of this project, joining discovery driven research to the applications of findings to crop improvement in horticulture. The educational events where research outputs were disseminated also included classroom settings and informal student-faculty interactions. PARTICIPANTS: Individuals who participated in this project include: Alan G. Smith, Principal Investigator who supervised all research and publication production as well as developed support and complementary funding for the project. Two faculty members of the University of Minnesota Department of Horticultural Science contributed to the research in interspecific incompatibility of Nicotiana and collaborations with Bielefeld University, Bielefeld, Germany and CABI Europe-Switzerland contributed to our research effort to understand plant invasiveness. An MS-level research associate in the department of Horticultural Science was trained in the areas of mutagenesis breeding for seedless plant production and interspecific incompatibility of Nicotiana. A Ph.D. student in the Applied Plant Sciences Graduate Program was trained in the areas of policy and regulation of genetically modified crops and reproductive biology of Carex species A second Ph.D. student in the Plant Biological Sciences Graduate Program focused on researching prezygotic interspecific barriers and speciation in Nicotiana was trained in several areas of biochemistry and molecular genetics. Three undergraduate students provide valuable assistance in research as well as having independent research projects in mutagenesis breeding for seedless plant production, plant tissue culture, gene introduction and DNA sequencing and analysis. The project has important partners in the horticultural industry and green industry trade organization with common interests and goals that provide direct and in-kind funding and collaboration to accomplish research support. TARGET AUDIENCES: The target audience for this project is a diverse audience of public and private groups. The research on interspecific incompatibility is of interest to geneticists, cell and plant reproductive biologists. The research also impacts private industry, specifically the green industry including ornamental, nursery and landscape plant producers, landscape design and installation and consumers. Efforts to provide information include formal classroom teaching in undergraduate and graduate course offerings. The most significant learning opportunities include an upper-level undergraduate course (Applications of Biotechnology to Plant Improvement) and a graduate level courses (Manipulation of Plant Growth and Reproduction, Research Ethics in the Plant and Environmental Sciences). Other effort occurs through publications and presentations. PROJECT MODIFICATIONS: No significant project modifications have been made during this reporting period.
Impacts
The goal of this project is to understand the molecular genetics of gene expression and how fundamental knowledge can be translated into plant improvement. Reproductive isolation is a key process in evolution and speciation that has contributed to the success and spread of terrestrial plants. It is also a barrier to the introgression of genes by interspecific hybridization. Interspecific incompatibility is the failure of reproductive events resulting in a block of hybrid production among taxa. An objective of this project was to identify the gene and proteins that regulate interspecific incompatibility in Nicotiana species. Toward this end, a novel semi-in vivo pollen tube growth assay was developed that was to identify key proteins that regulate the prezygotic interspecific incompatibility. The PELP was the major component in fractions with specific-inhibitory activity and N. tabacum plants with suppressed PELP accumulation lost inhibition of incompatible pollen tubes. It was concluded that PELP acts as a barrier to hybridization among some Nicotiana species and may be a speciation gene. Portions of the PELP gene were sequenced from a number of Nicotiana species to measure species-specific changes and to establish PELP phylogenetic relationships. In a project with the goal to produce noninvasive varieties of popular, but invasive landscape plants, high energy irradiation of seeds, rooted and unrooted stem cuttings from several woody perennial taxa was tested for the production of sterile plants. Survival of the plant material, growth rate, novel phenotypes, flowering and seed set is measured in the laboratory, greenhouse and field. Due to the protracted juvenile period of some of the woody perennial plant species, their growth, development and sexual reproductive competence is being assessed over several years. In project to understand the invasive nature of Tanacetum vulgare, the chemical and genetic diversity of invasive and native populations was compared. High genetic diversity as well as phenotypic plasticity of introduced plant populations may pose key advantages under changed selection pressures. However, a loss of genetic diversity can take place due to genetic bottlenecks, founder effects and inbreeding, which affect phenotypic traits such as plant chemistry. Tanacetum vulgare is of Eurasian origin, but was introduced for medical use to North America, where it became invasive. This project measured the genetic and chemical diversity of T. vulgare, populations. Based on Gaussian clustering and non-metric multidimensional scaling of these data it was concluded that there was high variation between and within populations. For T. vulgare plants of the invasive range, no loss of genetic diversity and a high chemical diversity was detected, which may be important traits for its successful spread.
Publications
- Wolf, V.C., A. Gassmann, B.M. Clasen, A.G. Smith, C. Muller. 2012. Genetic and chemical variation of Tanacetum vulgare in plants of native and invasive origin. Biological Control. 61:240-245.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Outputs from this project have been disseminated directly through refereed publications, local and national presentations, posters at professional society meetings, talks to funding and clientele groups, and educational outlets through classroom teaching and undergraduate and graduate student involvement in research. The outputs generated by this project were also indirectly disseminated through research grant proposals for continued and increased funding. These include research proposals to federal agencies, for example NSF and non-governmental agencies such as foundations and industry. Other means of dissemination include seminars, poster presentations and meeting with colleagues and collaborators at the University of Minnesota. The formal and informal reporting of research results builds a foundation for others to follow our progress and evaluate successes. Presentations made in 2011 include: McGinnis, E., A.G. Smith, M. Meyer. 2011. Analysis of U.S. Genetically Engineered Crop Regulation and Litigation. Microbial and Plant Genomics Institute Symposium. Changbin C., A.D. Farmer, R.J. Langley, J. Mudge, S. Dukowic-Schulze, G.D. May, J. Huntley, A.G. Smith and E.F. Retzel. Comparative transcriptomics of Arabidopsis stage 5-7 anthers and isolated male meiocytes. Plant and Animal Genome XX Conference. Eberle, C.A., B.M. Clasen, N. O. Anderson, A. D. Hegeman and A. G. Smith. 2011. Mechanisms and genes regulating Nicotiana interspecific incompatibility. Pollen Research Coordination Network Meeting, August 5-6 2011. Smith A.G. and E.E. McGinnis. 2011. Organizers for Plant Biotechnology Working Group Workshop "Regulation of Genetically Engineered Crops: Hawaii and Beyond." Annual Conference of the American Society for Horticultural Science, 26 September 2011. McGinnis, E. E., A. G. Smith and M. H. Meyer. 2011. Flower Induction in Carex pensylvanica Lam. Annual Meeting of the American Society of Plant Biologists, August 6, 2011. Eberle, C.A., N. O. Anderson, A. D. Hegeman and A. G. Smith. 2011. Mechanisms and genes regulating Nicotiana interspecific incompatibility. Annual Meeting of the American Society of Plant Biologists, August 6, 2011. In addition to traditional classroom teaching, undergraduates are actively involved in this research. Two undergraduates pursued independent research as well as provided valuable laboratory support. The development and delivery of educational material and seminar presentations represent the interdisciplinary nature of this project, joining discovery driven research to the applications of findings to crop improvement in horticulture. The educational events where research outputs were disseminated also included classroom settings and informal student-faculty interactions. For example, results from this project were presented to the horticulture industry at the Minnesota Green Expo. PARTICIPANTS: Individuals who participated in this project include: Alan G. Smith, Principal investigator who supervised all research and publication production as well as developed support and complementary funding for the project. Two faculty members of the University of Minnesota Department of Horticultural Science contributed to the research in interspecific incompatibility of Nicotiana. A Ph.D. graduate student and professor at Bielefeld University, Bielefeld, Germany and a professor at CABI Europe-Switzerland, Delemont, Switzerland contributed to our research effort to understand plant invasiveness. An MS-level research associate in the department of Horticultural Science was involved in research for sterility gene construction, transformation, mutagenesis breeding, sterile-plant evaluation and breeding and interspecific incompatibility of Nicotiana. A Ph.D. student in the Applied Plant Sciences Graduate Program began research in the areas of policy and regulation of genetically modified crops and reproductive biology of Carex spp. A second Ph.D. student in the Plant Biological Sciences Graduate Program is focused on researching prezygotic interspecific barriers and speciation in Nicotiana. A Ph.D., Research Assistant Professor collaborated on several projects involving meiosis and plant reproduction in Arabidopsis, sunflower, strawberry and tomato. Two undergraduate students provide valuable assistance in research as well as having independent research projects in genetic diversity and interspecific incompatibility. The project has important partners in the horticultural industry and green industry trade organization with common interests and goals that provide direct and in-kind funding and collaboration to accomplish research support. TARGET AUDIENCES: The target audience for this project is a diverse audience of public and private groups. The research on interspecific incompatibility is of interest to geneticists, cell and plant reproductive biologists. The research also impacts private industry, specifically the green industry including ornamental, nursery and landscape plant producers, landscape design and installation and consumers. Efforts to provide information include formal classroom teaching in undergraduate and graduate course offerings. The most significant learning opportunities include an upper-level undergraduate course (Applications of Biotechnology to Plant Improvement) and a graduate level course (Manipulation of Plant Growth and Reproduction). Other effort occurs through publications and presentations. PROJECT MODIFICATIONS: No significant project modifications have been made during this reporting period.
Impacts
The goal of this project is to understand the molecular genetics of gene expression and how fundamental knowledge can be translated into plant improvement. Reproductive isolation is a key process in evolution and speciation that has contributed to the success and spread of terrestrial plants. It is also a barrier to the introgression of genes by interspecific hybridization. Cell-cell interactions between pollen tubes and the pistil is a point at which mating among plant species is regulated and forms the basis for prezygotic reproductive barriers and species isolation. Interspecific incompatibility is the failure of reproductive events resulting in a block of hybrid production among taxa. An objective of this project was to identify the gene and proteins that regulate interspecific incompatibility in Nicotiana spp. Toward this end, a novel semi-in vivo pollen tube growth assay was developed that was to identify key proteins that regulate the prezygotic interspecific incompatibility. The pistil-specific extensin-like protein (PELPIII) was the major component in fractions with specific-inhibitory activity, implicating PELPIII as required for pollen tube growth inhibition. PELPIII was identified using PELPIII-specific antibodies in protein-gel blots and by analysis of QStar-Pulsar LC MS/MS peptide sequence. The requirement for N. tabacum PELPIII for the inhibition of N. obtusifolia pollen tube growth was supported by loss of specific-inhibitory activity in N. tabacum styles, which had reduced PELPII accumulation after antisense-PELPIII gene introduction. Therefore, PELPII is required for the interspecific incompatibility between N. tabacum and N. obtusifolia. This is the first assignment of function to the PELPIII protein. In a project with the goal to produce noninvasive varieties of popular, but invasive landscape plants, high energy irradiation of seeds, rooted and unrooted stem cuttings from several woody perennial taxa were tested for the production of sterile plants. Data was collected survival of the plant material after irradiation treatment. The level for most efficient production of sterile plants kills at least 50% of the material (the LD50). The LD50 is determined by counting the survivors of a specific radiation level relative to the number of survivors in the untreated controls. Data was also collected on height, growth habit and unusual phenotypes. Due to the protracted juvenile period of some of the woody perennial plant species, their growth, development and sexual reproductive competence will be assessed over several years.
Publications
- Smith A.G. and B.M. Clasen. 2011. Development of seedless taxa of popular invasive landscape plants. Combined Proceedings International Plant Propagators' Society 60: 69-77.
- Clasen, B.M, N.G. Moss, M.A. Chandler, A.G. Smith. 2010. Genetic Diversity Within and Among Populations of Common Tansy (Tanacetum vulgare). Canadian J. Plant Biology 91:717-723.
- Eberle, C.A., B.M. Clasen, N. O. Anderson and A. G. Smith. 2011. A novel pollen tube growth assay utilizing a transmitting tract-ablated Nicotiana tabacum style. Sexual Plant Reproduction. DOI 10.1007/s00497-011-0177-9.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Outputs from this project have been disseminated directly through publications, seminars and posters at professional society meetings, talks to funding and clientele groups, and educational outlets through classroom teaching and undergraduate and graduate students involvement in research. The outputs generated by this project were also indirectly disseminated through research grant proposals for continued and increased funding. These include research proposals to federal agencies, for example USDA and NSF and non-governmental agencies such as foundations and industry. A significant process in applying for federal and non-federal grants is the release of the information in refereed publications and other formats. Other formats include seminars, poster presentations and meeting with colleagues and collaborators. The formal and informal reporting of research results builds a foundation for others to follow our progress and evaluate successes. Presentations were made at: The 2010 Joint Annual Meeting of the American Society of Plant Biologists & the Canadian Society of Plant Physiologists/La Society Canadienne de Physiologie Vegetale, The XXI International Congress on Sexual Plant Reproduction, The Plant Biological Sciences Annual Retreat and its Orientation for Incoming Graduate Students, The Ohio Invasive Plant Research Conference: Connecting Research and Land Management, The 60th Annual Meeting of the IPPS Eastern Region, and the Minnesota-Wisconsin Invasive Species Conference, "Food for Thought" Lectures on Sustainable Ag and Biotechnology, Oregon State University, The meetings of collaborators for management of Tanacetum vulgare (common tansy), which included the University of Minnesota, Minnesota Department of Agriculture, the Bielefeld University, Germany; CABI E-CH, Switzerland, and McClay Ecoscience, Canada. In addition to traditional classroom teaching, a class on pollen tube growth and measurement of gene expression was taught at the Plant Biological Sciences Graduate Program's Summer Orientation at the Itasca Biological Station, MN. This non-traditional course provided incoming students a laboratory research experience in reproductive biology and molecular analysis of floral gene expression. These development and delivery of educational material and seminar presentations represent the interdisciplinary nature of this project, joining discovery driven research to the applications of findings to crop improvement in horticulture. The educational events where research outputs were disseminated also included classroom settings and informal student-faculty interactions. For example, results from this project were presented to the horticulture industry at the Minnesota Green Expo. PARTICIPANTS: Individuals who participated in this project include: Alan G. Smith, Principal investigator who supervised all research and publication production as well as developed support and complementary funding for the project. An MS-level research associate in the department of Horticultural Science was involved in research for sterility gene construction, transformation, mutagenesis breeding, sterile-plant evaluation and breeding and interspecific incompatibility of Nicotiana spp. A Ph.D. student in the Applied Plant Sciences Graduate Program began research in the areas of policy and regulation of genetically modified crops and reproductive biology of Carex spp. A second Ph.D. student in the Plant Biological Sciences Graduate Program is focused on researching prezygotic interspecific barriers and speciation in Nicotiana. A Ph.D., Research Assistant Professor collaborated on several projects involving meiosis and plant reproduction in Arabidopsis, sunflower, strawberry and tomato. Three undergraduate students provide valuable assistance in research as well as having independent research projects in genetic diversity and interspecific incompatibility. One of the undergraduates involved in this research program was awarded a fellowship from the World Food Prize Internship Program. Training and professional development opportunities were offered during weekly laboratory meetings, at formal classroom setting and through opportunities to travel to professional meetings and to other laboratories for training and collaboration. The project partners with the horticultural industry and green industry trade organization to establish common interests and goals and accomplish research through intellectual and in-kind support. TARGET AUDIENCES: The target audience for this project includes graduate and undergraduate students, post-doctoral graduates and professional staff. In addition the research impacts private industry, specifically the green industry including ornamental, nursery and landscape plant producers and consumers. Efforts to provide information include formal classroom teaching in undergraduate and graduate course offerings. The most significant learning opportunities include an upper-level undergraduate course (Applications of Biotechnology to Plant Improvement) and a graduate level course (Manipulation of Plant Growth and Reproduction). Other effort occurs through publications and presentations. PROJECT MODIFICATIONS: No significant project modifications have been made during this reporting period.
Impacts
The goal of this project is to understand the molecular genetics of gene expression and how fundamental knowledge can be translated into plant improvement. Reproductive isolation is a key process in evolution and speciation that has contributed to the success and spread of terrestrial plants. Cell-cell interactions between pollen tubes and the pistil is a point at which mating among plant species is regulated and forms the basis for prezygotic reproductive barriers and species isolation. Interspecific incompatibility is the failure of reproductive events resulting in a block of hybrid production among taxa. An objective of this project is to identify the gene and proteins that regulate interspecific incompatibility in Nicotiana spp. Toward this end, a novel semi-in vivo pollen tube growth assay, was developed. The salient features of this assay include normal pollen tube growth, sustained growth rates and normal pollen tube morphology through several centimeters of growth in the style. The pollen tube growth assay has the advantage that growth conditions and environmental factors can be controlled and specific treatments or protein preparations can be added directly to pollen tubes to measure their effect on pollen tube growth. The assay provides a method to follow the specific activity of key proteins that regulate the interspecific incompatibility reaction. To identify key proteins that regulate the prezygotic interspecific incompatibility, several of the most abundant proteins from fractions having specific activity toward the inhibition of interspecific pollen tube growth derived from the cation exchange chromatography were subjected to LC MS/MS. Analysis of these results are ongoing and should lead to the identification of candidate genes involved in the interspecific incompatibility mechanism. To model gene action in interspecific incompatibility crosses where made among several Nicotiana spp. to determine their relative interspecific compatibility and to derive F1 seed, where possible. The interspecific incompatibilities of the F1 plants will be measured to determine how species genomes interact in this complex process. The F1 individuals will be crossed or selfed to generate F2 populations that will be used to model genetic relationships and inheritance of specific interspecific interactions. In a project to eliminate landscape plan invasiveness, both biotechnology and mutation breeding strategies are being used. This project occurs in collaboration with the horticultural industry. High energy irradiation of seeds, rooted and unrooted stem cuttings from several woody cultivars were tested for the production of sterile plants. Data was collected on height, growth habit and unusual phenotypes. Due to the protracted juvenile period of some of the woody perennial plant species, their growth, development and sexual reproductive competence will be assessed over several years.
Publications
- Eberle, C.A., N. O. Anderson, A. D. Hegeman and A. G. Smith. 2010. XXIst International Congress on Sexual Plant Reproduction, August 6, 2010.
- Smith, A G. 2010. Horticultural Plant Improvement at the University of Minnesota. International Plant Propagators Society Eastern Region, September 29, 2010.
- McGinnis, E. E., M. H. Meyer and A. G. Smith. 2010. Sweet and Sour: A Scientific and Legal Look at Herbicide-Tolerant Sugar Beet. Plant Cell 22: 1653-1657.
- Clasen, B., and A.G. Smith. 2010. Development of Non-invasive Plant Alternatives for Use in the Landscape. Minnesota-Wisconsin Invasive Species Conference, November 10, 2010.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Outputs from this project have been disseminated directly through publications, seminars and posters at professional society meetings, talks to funding and clientele groups, educational outlets and indirectly through grant proposals. Some of the output generated by this project is indirectly disseminated through research grant programs for continued and increased funding. These include grants to federal agencies, for example USDA and NSF and non-governmental agencies such as foundations and industry. A significant process in applying for non-federal grants is the description of the current research program status and its fit with the goals and objects of the foundation or industry. This involves reporting on our strategy, results and conclusions to selection committees or industry leaders. Additionally, presentations were made at: The 9th International Plant Molecular Biology meeting; the meeting of the 59th Annual Meeting of the IPPS Eastern Region; The Cochran Fellowship training program, sponsored by Center for Regulatory Research LLC and the USDA; a meetings of collaborators for biocontrol of Tanacetum vulgare (common tansy), which included the Minnesota Department of Agriculture, the Commonwealth Agricultural Bureaux International (CABI, Switzerland), McClay Ecoscience, Canada. In addition to traditional classroom teaching, a class on pollen tube growth and measurement of gene expression was taught at the Plant Biological Sciences Graduate Program's Summer Orientation at the Itasca Biological Station, MN. This non-traditional course provided incoming students a laboratory research experience in reproductive biology and molecular analysis of floral gene expression. A thesis proposal was presented to the Applied Plant Sciences graduate program with the title "Will GMOs become an endangered species within the sustainable landscape" and a defense seminar was presented to the same group with the title "Weeds Today, Gone Tomorrow". These presentations, courses and lectures represent the interdisciplinary nature of this project, joining discovery driven research to the applications of findings to crop improvement in horticulture. The educational events where research outputs were disseminated also included classroom settings and informal student-faculty interactions. For example, results from this project were presented during informal presentations, during recruitment, as a part of new student orientation activities and during informal presentations to the industry at the Minnesota Green Expo. Outcomes and impacts disseminated through publications are discussed in the next section. PARTICIPANTS: Individuals who worked on this project include: Alan G. Smith, Principal investigator who supervised all research and publication production as well as developed support and complementary funding for the project. An MS-level research associate in the department of Horticultural Science was involved in research for sterility gene construction, transformation, mutagenesis breeding, sterile-plant evaluation and breeding and interspecific incompatibility of Nicotiana spp. An M.S. degree was granted from the Applied Plant Sciences Graduate Program in the area genetic diversity of an invasive weed and reducing non-native plant invasiveness. A Ph.D. student in Applied Plant Sciences Graduate Program began research in the areas of policy and regulation of genetically modified crops and reproductive biology of Carex. A second Ph.D. student in the Plant Biological Sciences Graduate Program is focused on research prezygotic interspecific barriers and speciation in the broader areas of interspecific incompatibility in Nicotiana. A Ph.D., Research Assistant Professor collaborated on several projects involving meiosis and plant reproduction in Arabidopsis, sunflower, strawberry and tomato. Three undergraduate students provide valuable assistance in research as well as having independent research projects in interspecific incompatibility in Nicotiana spp. and self-incompatibility in N. alata. One undergraduate was awarded a grant from the University of Minnesota's Undergraduate Research Opportunities Program to study pollen-pistil interactions in several related species of Nicotiana. Training and professional development opportunities were offered during weekly laboratory meetings, at formal classroom setting and through opportunities to travel to professional meetings and to other laboratories for training and collaboration. The project partners with the horticultural industry and green industry trade organization to establish common interests and goals and accomplish research through intellectual and in-kind support. TARGET AUDIENCES: The target audience for this project includes graduate and undergraduate students, post-doctoral graduates and professional staff. In addition the research impacts private industry, specifically the green industry including ornamental, nursery and landscape plant producers and consumers. Efforts to provide information include formal classroom teaching in undergraduate and graduate course offerings. The most significant learning opportunities include an upper-level undergraduate course (Applications of Biotechnology to Plant Improvement) and a graduate level course (Manipulation of Plant Growth and Reproduction). Other effort occurs through publications and presentations. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The goal of this project is to understand the molecular genetics of gene expression and how fundamental knowledge can be translated into plant improvement. Reproductive isolation is a key process in evolution and speciation that has contributed to the success and spread of terrestrial plants. Cell-cell interactions between pollen tubes and the pistil is a point at which mating among plant species is regulated and forms the basis for prezygotic reproductive barriers and species isolation. Interspecific incompatibility is the failure of reproductive events resulting in a block of hybrid production among taxa. An objective of this project is to identify the gene and proteins that regulate interspecific incompatibility in Nicotiana spp. Toward this end, a novel semi-in vivo pollen tube growth assay, was developed. This assay provides a method to follow the specific activity of key proteins that regulate the interspecific incompatibility reaction. In a second project the model genetic plant Arabidopsis thaliana is being used to identify key genes in meiosis and understand how they are regulated. Meiosis is essential for genetic recombination and gamete formation and is a critical process in the life cycle of all eukaryotes with sexual reproduction. Transcripts were extracted from A. thaliana meiocytes and Solexa/Ilumina transcriptome sequencing performed to identify mRNAs with higher accumulation in anthers and meiocytes relative to seedling tissues. Many novel genes were identified are being characterized using loss of function mutations from T-DNA knockouts. In a third project, both biotechnology and mutation breeding strategies are being used for the production of sterile and therefore non-invasive cultivars of invasive horticultural plants. This project occurs in collaboration with the horticultural industry and has the objective to eliminate invasiveness of horticultural crops. Toward this objective, irradiation of seeds, rooted and unrooted stem cuttings from several woody cultivars were completed. Data was collected on height, growth habit and unusual phenotypes. Due to the protracted juvenile period of some of these plants species, their growth, development and sexual reproductive competence will be assessed over several years.
Publications
- McNeil, K.J., and A.G. Smith. 2009. A glycine-rich protein that facilitates exine formation during tomato pollen development. Planta, accepted.
- Eberle, C., N.O. Anderson and A.G. Smith. 2009. Novel biochemical approach to identification of interspecific incompatibility factors involved in pollen tube growth inhibition.9th International Plant Molecular Biology, October 25-30, 2009.
- Chen C., A. Farmer , G.D. May, A.G. Smith, J. Huntley, N. Miller, J. Mudge, E.F. Retzel. 2009. Meiotic transcriptome analysis in Arabidopsis. Plant & Animal Genomes XVII Conference.
- Clasen, B. 2009. Genetic Structure of Common Tansy Populations using Inter-simple Sequence Repeat Markers and Plant Improvement through Gamma Radiation Mutagenesis Breeding. Master's Thesis, University of Minnesota.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Outputs from this project have been disseminated directly through publications, seminars and posters at professional society meetings, talks to funding and clientele groups, educational outlets and indirectly through grant proposals. Some of the output generated by this project is indirectly disseminated through research grant programs for continued and increased funding. These include grants to federal agencies, for example USDA and NSF and non-governmental agencies such as foundations and industry. A major part of the application process, especially for non-federal sources and industry is the description of the current research program status and its fit with the goals and objects of the foundation or industry. This may require description of results in written reports or presentation to selection committees. Four presentations were made at professional society meetings. These include meetings of the Joint Annual Meeting of the American Society of Plant Biologists and the Sociedad Mexicana De Bioquimicaand, the American Society of Horticultural Science and the Minnesota Invasive Species Conference. In addition, outputs were presented at meetings with specific scientific objectives. The first of these meetings included international collaborators from Minnesota Department of Agriculture (MDA), the Commonwealth Agricultural Bureaux International (CABI) and McClay Ecoscience. This meeting was to report and strategize on the funding and research for development of biocontrol agents for tansy. This project reported on research results that measured the genetic diversity of invasive populations of Tanacetum vulgare. The others were thesis proposals presented to the Plant Biological Sciences and Applied Plant Sciences graduate programs. The educational events where research outputs were disseminated include classroom settings and informal student-faculty interactions. For example, results from this project were presented in a freshman class to demonstrate faculty research activities from the Department of Horticultural Science, as a part of an undergraduate class introducing plant genetics and biotechnology. Results were also presented as part of a graduate class on breeding of asexually propagated crops. Less formal presentations of outputs include recruitment and orientation activities and informal presentations to the industry at the Minnesota Green Expo. Outcomes and impacts disseminated through publications are discussed in the next section. PARTICIPANTS: Individuals who worked on this project include: Alan G. Smith, Principal investigator who supervised all research and publication production as well as developed support and complementary funding for the project. Nicole Gardner, research associate in the department of Horticultural Science was involved in research for sterility gene construction, transformation, sterile and rolC transgenic plant evaluation and breeding and interspecific incompatibility of Nicotiana spp. Ben Clasen, Applied Plant Sciences graduate student began research toward and MS degree using tissue culture micropropagation to develop systems for adventitious shoot formation and gene transfer in Buddleia spp. and assessing Tanacetum vulgare genetic diversity. Carrie Eberle, Plant Biological Sciences Ph.D. graduate student was focused on speciation and interspecific incompatibility in Nicotiana. Dr. Changbin Chen, Research Assistant Professor collaborated on several projects involving meiosis and plant reproduction in Arabidopsis, sunflower, strawberry and tomato. Jason Skinner an undergraduate student in the major of Environmental Horticulture was a laboratory assistant providing general lab help and care for greenhouse grown plants. Jake Lau was trained for route tissue culture media preparation and care of plants in culture and was responsible for field testing of genetically modified plants. Mr. Lau was also funded by a grant from the University of Minnesota's Undergraduate Research Opportunities Program to study genetic diversity of European and North American Tanacetum populations. Training and professional development opportunities were offered during weekly laboratory meetings, at formal classroom setting and through opportunities to travel to professional meetings and to other laboratories for training and collaboration. TARGET AUDIENCES: The target audience for this project includes graduate and undergraduate students, post-doctoral graduates and professional staff. In addition the research impact private industry, specifically the green industry including ornamental, nursery and landscape plant producers and consumers. Efforts to provide information include formal classroom teaching in undergraduate and graduate course offerings. The most significant learning opportunities include an upper-level undergraduate course (Applications of Biotechnology to Plant Improvement) and a graduate level course (Manipulation of Plant Growth and Reproduction). Other effort occurs through publications and presentations. PROJECT MODIFICATIONS: No significant project modifications have been made during this reporting period.
Impacts
The goal of this project is to understand the molecular genetics of gene expression and how fundamental knowledge can be translated into plant improvement. Reproductive isolation is a key process in evolution and speciation that has contributed to the success and spread of terrestrial plants. Cell-cell interactions between pollen tubes and the pistil is a point at which mating among plant species is regulated and forms the basis for prezygotic reproductive barriers and species isolation. Interspecific incompatibility is the failure of reproductive events resulting in a block of hybrid production among taxa. An objective of this project is to identify the proteins that regulate interspecific incompatibility in Nicotiana spp. Toward this end a novel semi-in vivo pollen tube growth assay, was developed. This assay provides a method to follow the specific activity of key proteins that regulate the interspecific incompatibility reaction. In a second project the model genetic plant Arabidopsis thaliana is being used to identify the key genes in meiosis and understand how they interact and how the processes of meiosis are regulated. Meiosis is essential for genetic recombination and gamete formation and is a critical process in the life cycle of all eukaryotes with sexual reproduction. We have developed a novel, robust, and efficient method of Capillary Collection of Meiocytes. This method permits collection of meiocytes in quantities sufficient for mRNA extraction and analysis by microarray and Solexa/Ilumina transcriptome sequencing. In a third project, both biotechnology and mutation breeding strategies are being used for the production of sterile and therefore non-invasive cultivars of invasive horticultural plants. This project occurs in collaboration with the horticultural industry and has the objective to eliminate invasiveness of horticultural crops. Toward this objective, irradiation of seeds, rooted and unrooted stem cuttings from several woody cultivars were completed. Data was collected on height, growth habit and unusual phenotypes. Several invasive crop plants were initiated in culture for use in sterility gene introduction. In a fourth project to utility a well characterized dwarfing gene from Agrobacterium rhizogenes, the dwarfing gene was introduced into the ornamental plant, Nicotiana sylvestris. The first field trial of genetically modified ornamental Nicotiana sylvestris were completed. Plant growth, flowering, architecture and aesthetics were evaluated.
Publications
- Gardner, N., R. Felsheim and A.G. Smith. 2008. Production of male- and female-sterile plants through reproductive tissue ablation. J. Plant Physiol. In press.
- Smith, A.G., N. Gardner and B. Clasen. Eliminating invasiveness of horticultural plants. 2008. The Scoop 31 (7): 64-65. B. Clasen, N. Gardner and A.G. Smith. 2008. Reducing the Invasive Potential of Horticultural Crops. Yard & Garden News, 10 (17), see http://www.extension.umn.edu/gardeninfo/index.html.
- Smith, A.G. 2008. Biotechnology Workshop: Emerging Technologies for Biotechnology and Crop Improvemnt. HortScience 43(4): 1056.
- B. Clasen, A.G. Smith and N. Gardner. October 26-29, 2008. Genetic diversity among and between populations of common tansy (Tanacetum vulgare). Minnesota Invasive Species Conference.
- A.G. Smith, N. Gardner and B. Clasen. October 26-29, 2008. Elimination of invasiveness in horticultural crops through biotechnology. Minnesota Invasive Species Conference.
- Smith, A.G. 2008. Colloquium: Systems Biology for Horticultural Sciences and How to Participate. 43(4): 1052.
- Smith, A.G., C. Chen, E.F. Retzel and J.J. Huntley. 2008. Application of Whole Transcriptome Shotgun Sequencing. HortScience 43(4): 1056.
- Eberle, C., N. Gardner, N.O. Anderson and A.G. Smith. 2008. The Transmitting Tract of Nicotiana tabacum Inhibits Interspecific Pollen Tube Growth of N. obtusifolia.Joint Annual Meeting of the American Society of Plant Biologists and the Sociedad Mexicana De Bioquimica. June 26 - July 1, 2008. P35019.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Outputs from this project have been disseminated indirectly through grant proposals and directly through publications, seminars and posters at professional society meetings, talks to clientele groups, and educational outlets. Some of the output generated by this project is indirectly disseminated through research grant programs for continued and increased funding. These include grants to federal agencies, for example USDA and NSF and non-governmental agencies such as foundations and industry. A major part of the application process, especially for non-federal sources and industry is the description of the current research program and its fit with the goals and objects of the foundation or industry. This may require description of results in written reports or presentation to selection committees. Two presentations were made at professional society meetings. These include the joint meeting of the American Fern Society, American Society of Plant Biologists, American Society of
Plant Taxonomists and Botanical Society of America and another society meeting of the American Society of Horticultural Science. In addition, outputs were presented at two meetings of special interest groups. The first included a group interested in risk analysis of invasive plants and animals and was titled Tanacetum vulgare (common tansy) invasion of North America. The second involved a group of scientist interested in reproductive biology in plants and fungi at the University of Florida that was titled interspecific incompatibility in Nicotiana spp. The educational events where research outputs are disseminated include classroom settings and informal student-faculty interactions. For example, results from this project were presented in a freshman class to demonstrate faculty research activities from the Department of Horticultural Science and as a part of a class introducing plant genetics and biotechnology. Less formal presentations of outputs include recruitment and orientation
activities such as the College of Food, Agriculture and Natural Resources Science Deans' welcome reception or presentations to the industry such as at the 2007 Minnesota Green Expo. Outputs disseminated through publications are discussed in the next section.
PARTICIPANTS: Participants in this project include: Alan G. Smith, principal investigator who directs and does research as well as most of the outreach and dissemination of project outcomes. Nicole Gardner, research associate receives partial salary and has contributed to all aspects of the project in doing and documenting research activities. Benjamin Clasen is an undergraduate laboratory assistant who contributed to research on biotechnology improvement of horticultural crops and understanding traits that contribute to the invasiveness of Tanacetum vulgare. Partner organizations include Bailey Nurseries, Minnesota Nursery and Landscape Association, J. Frank Schmidt Family Charitable Foundation, The Office of the Vice President for Research, Minnesota Futures Grant Program, Microbial and Plant Genomics Institute of the University of Minnesota. Collaborators include: Carrie Eberle, Plant Biological Science graduate student, Neal Anderson, Associate Professor in the Department of
Horticultural Sciences, and Changbin Chen, Research Assistant Professor in the Department of Horticultural Sciences. Training involved three undergraduates from the University of Minnesota that were directly involved in research activities.
TARGET AUDIENCES: The applied aspects of this project are directed toward the green industries, specifically the landscape and nursery sections. Improved plants provide a more sustainable industry, greater growth potential and increased incomes. The public will benefit directly from plant materials with increased aesthetics, sustainability and reduced environmental impacts. The public also benefits from developing new technologies, such as gene transfer and understanding reproduction in plants that can contribute to broad areas of agriculture and horticulture. Efforts to deliver science-based knowledge include involving undergraduates directly in project research, classroom teaching in the areas of plant growth and development, genetics and biotechnology and through industry contacts such as discussion with local industry and activities during events such as the Green Expo run by the Minnesota Nursery and Landscape Association.
Impacts
The goal of this project is to understand the molecular genetics of gene expression and how fundamental knowledge can be translated into plant improvement. Reproductive isolation is a key process in evolution and speciation that has contributed to the success and spread of terrestrial plants. Cell-cell interactions between pollen tubes and the pistil is a point at which mating among plant species is regulated and forms the basis for prezygotic reproductive barriers and species isolation. Interspecific incompatibility (II) is the failure of reproductive events resulting in a block of hybrid production among taxa. An objective of this project is to determine the mechanisms of prezygotic barriers to fertilization that regulate II in Nicotiana spp. In this project we focus on the TT as the pistil tissue essential for II. The TT provides a pathway from the stigma to the ovules where fertilization occurs. The TT was thought to be essential to pollen tube growth because of
the nutrients and guidance it provides to the pollen tubes. Our data demonstrates the II interactions occur within the TT of Nicotiana spp. To test the hypothesis that the mature TT of N. tabacum pistils is essential for II and pollen tube growth, pistils lacking mature TT were produced by introducing a gene that ablates the TT. These plants did not have a mature TT in the styles and necrosis of the stigma. In order to circumvent the necrotic stigma, stigmas were removed and styles were directly pollinated after application of a drop of the triacylglyceride, Trilinolein. Despite the consensus in the literature that the TT is essential for pollen tube growth, pollen tubes grew in the styles of N. tabacum that lacked a TT. The lack of a mature TT did not contribute to female infertility. To test whether the TT was essential for II, wildtype and TT ablated styles were pollinated with compatible and incompatible Nicotiana spp. pollen donors. Pollinations with some incompatible species
were converted to compatible when the TT was absent. Therefore, the TT is essential for II of these species. The conversion of II to interspecific compatible with the ablation of the TT provides a novel tool to identify the factor(s) produced by the N. tabacum pistil that stop pollen tube growth of incompatible species. High temperature stress imposed during the cell division stage of maize (Zea mays L.) kernel development adversely affects growth and mature mass. The mechanism by which heat stress affects early zein accumulation in maize kernels was researched by analyzing transcription rates of zein genes. The results indicated that the effects of heat stress on zein transcription rates are most likely affected by a delay in endosperm development.
Publications
- Carson, T.D., D.B. White and A.G. Smith. 2007. Distinguishing creeping bluegrass (Poa annua var. reptans) genotypes using inter-simple sequence repeat markers. HortScience 42:373-377. Eberle, C., N. Gardner and A.G. Smith. 2007. The Role of the Transmitting Tract in Pollination and Interspecific Incompatibility in Nicotiana. Plant Biology & Botany meeting July 7-11, 2007 P28015. Smith, A.G. 2007. Biotechnology Workshop: Biotechnology strategies for improving horticultural crops for human health. HortScience 42(4): 791.
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Progress 01/01/06 to 12/31/06
Outputs
The goal of this project is to understand the molecular genetics gene expression. High temperature stress imposed during the cell division stage of maize (Zea mays L.) kernel development adversely affects growth and mature mass. The mechanisms by which heat stress affects early zein accumulation in maize kernels was researched. Both the 27 kD and cluster 1 zeins of sub-family 4 (ZSF4C1) zein mRNA steady-state levels were significantly delayed by heat stress. Transcription rates of both zeins were reduced in the endosperm of kernels exposed to 4 DHS. These data indicates that the effects of heat stress on zein transcription rates are most likely affected by a delay in endosperm development. cDNAs of two biosynthetic genes in the anthocyanin pathway, leucoanthocyanidin dioxygenase (LDOX) and UDP glucose: flavonoid 3-O- glucosyl transferase (UFGT) that are responsible for the synthesis of red pigmentation of potato were cloned. The mRNA accumulation of these genes in
periderm during the development of tubers was determined using RNA gel blot analyses. LDOX RNA was detected in cortex tissue and microtubers, while UFGT RNA was detected in cortex tissue and periderm of young tubers, but not in periderm of tubers weighing more than 25 g. This suggests that accumulation of LDOX and UFGT mRNA may limit anthocyanin synthesis as 'Norland' tubers develop. The effects of differential rolC gene expression on plant height, leaf color, root growth, leaf size, corolla length, and stem diameter was determined. Differential expression of rolC in Nicotiana tabacum L. 'Samsun' plants was achieved using the 35S promoter, the light inducible rbcS promoter, or the native rolC promoter. Plants expressing rolC showed a wide range of phenotypes, with the largest changes in plants expressing rolC using the 35S promoter, which also had the highest rolC mRNA levels. Plants expressing rolC with the rolC or rbcS promoter had significant changes for many measured traits,
despite rolC mRNA levels that were not significantly different from non-transformed controls. As rolC mRNA levels increased, so did the severity of the rolC phenotype observed. Transformation with rolC can be useful in ornamental crops where smaller cultivars are desired. Manipulation of flower-specific gene expression can produce male- and female-sterile cultivars of plants, which are beneficial because of their increased potential for vegetative growth, increased flower number and longevity, prevention of gene flow, decreased invasiveness, decreased allergen production, and elimination of nuisance fruit. Sterile Petunia plants were shown to have increased flower life, a very important character for floriculture crops. We are using similar sterility gene constructs to eliminate the invasive potential of several horticultural crops.
Impacts
The study of gene expression can lead to direct improvements in horticultural crop characters including: ornamental value, plant invasiveness, and gene flow from genetically modified plants. This research project will increase the understanding of the function of genes in reproduction, and mechanisms that control organ-specific expression. Understanding the gene expression will alos determine how plant species are evolved and maintatined. These data are used in applications to produce plants with reduced size, reduced invasiveness, and other improved horticultural traits.
Publications
- Monjardino, P., Smith, A. G. and Jones, R. J. 2006. Zein transcription and endoreduplication in maize endosperm are differentially affected by heat stress. Crop Science, 46: 2581-258.
- Keifenheim, D.L., Smith, A. G. and Tong, B.S.. 2006. Cloning and expression of anthocyanin biosynthesis genes in developing tubers. Amer. J. Pot. Res. 83: 233-239.
- Gardner, N., Melberg, T., George, M. and Smith, A.G. 2006. Differential expression of rolC results in unique plant phenotypes. J. Amer. Soc. Hort. Sci. 131: 82-88.
- N. Gardner, and Smith, A. G. 2005. Producing smaller varieties of landscape plants at the University of Minnesota. Minnesota Nursery and Landscape Association (MNLA) Bulletin, 1: 26-27.
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Progress 01/01/05 to 12/31/05
Outputs
The goal of this project is to understand the molecular genetics of flower development and gene expression. Flower development and fertilization is a prerequisite for the formation of seeds and fruits of most of the economically important agronomic and horticultural plants, and is very important for ornamental plants. Reproduction is a major determinant of a plant's invasiveness and the route of gene flow from genetically modified plants. Invasive plants and the spread of transgenes can have major negative impacts on natural and cultivated ecosystems. The objectives of this project are to elucidate the functions of flower-specific genes, to develop improved horticultural crops, and to train students and other researchers. To understand the function of a small family of glycine-rich proteins (GRP) that accumulate specifically in the anther and pollen of tomato, the post-translational processing of the proteins were examined and the tomato genes were used to identify
similar genes in the model genetic plant, Arabidopsis thaliana. GRP 127 was shown to undergo a complex multi-step processing during pollen development. We have identified over 200 genes that encode GRPs in the Arabidopsis genome. Sequence analysis and expression data are being used to organize these genes into related groups to dissect their role in plant growth and development. Manipulation of flower-specific gene expression can produce male- and female-sterile cultivars of plants, which are beneficial because of their increased potential for vegetative growth, increased flower number and longevity, prevention of gene flow, decreased invasiveness, decreased allergen production, and elimination of nuisance fruit. The project's male- and female-sterility gene constructs have proven effective in Arabidopsis, Petunia and Nicotiana. Sterile Petunia plants were shown to have increased flower life, a very important character for floriculture crops. Controlling plant growth is important for
shipping and cultivation. Three gene constructs that express the Agrobacterium rhizogenes rolC gene in differing amounts and in different tissues were introduced into Nicotiana. Expression of the rolC gene reduced plant size overall and resulted in several novel plant phenotypes. Plants carrying the introduced rolC gene were backcrossed to non-transformed controls and the progeny were evaluated for inheritance of ornamental and landscaping qualities. Several N. alata, N. fructicosa, and N. sylvestris were produced with decreased plant height, altered leaf shape and size, reduced fertility, and near normal sized flowers. These will be trialed further to determine their performance in cultivation. Biotechnology approaches to crop improvement rely on the ability to introduce genes into plants, however, for many horticultural crops, methods for gene introduction are not available. Therefore, we will also develop and test strategies for gene introduction into plants that would benefit
greatly from reduced size or sterility. We have developed a method for gene introduction into the Euonymus alatus. This is essential for the introduction of sterility genes to eliminate E. alatus invasive potential.
Impacts
Flower development is important for crop yield, a primary character of ornamental value, a major determinant of a plant's invasiveness, and is the route for gene flow from genetically modified plants. This research project will increase the understanding of stamen and pistil development, the function of genes in reproduction, and mechanisms that control organ-specific expression. These data are used in applications to produce plants with reduced size, reduced invasiveness, and other improved horticultural traits.
Publications
- Smith, A.G. 2005. Tissue culture and gene introduction for nursery crop improvement. HortScience 40: 982.
- Smith, A.G. 2005. Challenges and strategies to fund horticultural biotechnology. HortScience 40: 934.
- Smith, A.G., N. Gardner and E. Zimmermann. 2005. Engineering female sterility for horticultural crops. HortScience 40: 1020.
- Smith, A.G., and E. Zimmermann. 2005. Adventitious shoot production and transformation of Euonymus alata. HortScience 40: 1081.
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Progress 01/01/04 to 12/31/04
Outputs
The goal of this project is to understand the molecular genetics of flower development and gene expression. Flower development is a prerequisite for the formation of seeds and fruits of most of the economically important agronomic and horticultural plants and is very important for ornamental plants. The objectives of this project are to elucidate the function and mechanisms that control flower-specific gene expression, to apply the understanding of the molecular genetics of flower development toward improving horticultural crops, and to train students and other researchers. To understand the function of the glycine-rich proteins in anther and pollen development in tomato an antibody to the glycine-rich protein (GRP) 127 was developed and used to show the developmental and organ and cell-specific accumulation of this protein. We have shown that the stamen-specific GRPs are developmentally processed and accumulate in tomato pollen. The project is also analyzing all GRPs
expressed in Arabidopsis thaliana. Data from the Arabidopsis genome project has been used to identify and analyze the GRP genes. These data will provide a better understanding of the function of these proteins in all plants. Male- and female-sterile cultivars of horticultural crops are beneficial by increasing vegetative growth, increasing flower number and longevity, preventing gene flow, decreasing invasiveness, decreasing allergen production, and eliminating nuisance fruit development. Sterile plants are produced using reproductive-specific promoters to ablate cells essential to female and male reproductive tissues. Male- and female-sterility gene constructs are being tested in Arabidopsis, Petunia and Nicotiana. There is increased demand for smaller, more compact plants in the nursery industry due to decreased space for growing plants in the landscape. Three gene constructs that express the Agrobacterium rhizogenes rolC gene in differing amounts and in different tissues were
introduced into Nicotiana to test their usefulness in producing smaller landscape plants with increased branching and unique phenotypes. Plants regenerated from tissue culture that contain the rolC gene constructs showed a variety of phenotypes. Overall, plants were shorter and had increased branching. Among plants carrying a specific construct the phenotypes varied significantly and variation was greatest when plants carrying different constructs were compared. Two of these constructs were introduced into ornamental tobacco (N. alata, N. fructicosa, and N. sylvestris. We are evaluating and crossing these lines with the goal of introducing a sterile-ornamental tobacco. Biotechnology approaches to crop improvement rely on the ability to introduce genes into plants, however, for many plants, methods for gene introduction are not available. Therefore, we will also develop and test strategies for gene introduction into landscape plants. These methods will be very useful to other
researchers wishing to improve plants through gene introduction.
Impacts
Plant reproduction is an essential step in the development of seeds and fruits and is a major determinant of ornamental value. This research project will increase the understanding of stamen and pistil development, the function of genes in reproduction, and mechanisms that control organ-specific expression. This information and other basic information on gene action has application to producing improved plants for the horticulture industry and understanding basic processes in plant development.
Publications
- Monjardino, P., A. G. Smith, and R. J. Jones. 2004. Effects of Heat Stress on Protein Accumulation of Maize (Zea mays L.) Kernels. In press: Crop Science.
- McNeil, K.J., E. and A.G. Smith. 2004. An anther-specific cysteine-rich protein of tomato localized to the tapetum and microspores. In Press: J. Plant Physiol.
- Smith, A.G., N. Gardner, and T. Melberg. 2004. Differential Expression of rolC Results in Unique Plant Phenotypes. HortScience 39: 756.
- Smith, A.G., and E. Zimmermann. 2004. Increased Flower Longevity in Petunia with Male Sterility. HortScience 39: 822.
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Progress 01/01/03 to 12/31/03
Outputs
The goal of this project is to understand the molecular genetics of flower development and gene expression. Flower development is a prerequisite for the formation of seeds and fruits of most of the economically important agronomic and horticultural plants and is a primary character of ornamental plants. The objectives of this project are to elucidate the function and mechanisms that control flower-specific gene expression, to apply an understanding of the molecular genetics of flower development toward improving horticultural crops, and to train students and other researchers. Two genes encoding glycine-rich proteins (GRPs) with stamen-specific expression are being characterized. The 92-GRP was determined to be localized in the outer wall of the pollen, the tapetum, and the orbicules that line the wall of the pollen sac. Plants with reduced 92-GRP expression have altered morphology of the outer pollen wall and reduced pollen viability. The 92-GRP is important for
uniform development of the pollen exine and pollen viability. It may function to facilitate the movement or deposition of sporopollenin on the pollen outer wall. Plants with reduced expression of a second gene, 127-GRP are being characterized. An antibody to the 127-GRP is being developed to assess gene expression and localize this protein during anther development. Petunia lines carrying a male-sterility gene construct and tobacco carrying a male- and female-sterility gene constructs were characterized. Petunias were completely male-sterile as determined by microscopy and seed set. Surprisingly, in greenhouse experiments these plants showed flower longevity twice that of male-fertile lines. Experiments will test the use of these constructs to increase flower longevity, eliminate nuisance fruit production, decrease invasiveness, reduce allergenic pollen production, increase flower number and flowering cycles, and prevent gene flow. The tobacco were male-sterile and partially female
fertile. Alterations in the sterility gene construct are being produced and tested for ability to confer female-sterility. There is increased demand for smaller, more compact plants in the nursery industry due to decreased space for growing plants in the landscape. Many trees, shrubs, and herbaceous plants are too large for the majority of residential landscapes. To test a biotechnology approach to reducing plant size, three related, but distinct, dwarfing-gene constructs that use the rolC gene were introduced into tobacco. The dwarfing genes used three different promoter sequences to produce different levels of the rolC gene product in different organs of the plant. The dwarfing genes produced remarkable variation in height, internode length, leaf size and shape, branching, flower morphology, and fertility relative to normal plants. The level of rolC gene expression is being determined to correlate gene expression with specific phenotypes. Theses data will be very useful in selecting
a specific gene for tailoring plant architecture, size, and leaf shape.
Impacts
Plant reproduction is an essential step in the development of seeds and fruits and is a major determinant of ornamental value. This research project will increase the understanding of stamen and pistil development, the function of genes in reproduction, and mechanisms that control organ-specific expression. This information and other basic information on gene action has application to producing improved plants for the horticulture industry and understanding basic processes in plant development.
Publications
- Bucciaglia, P.A., Zimmermann, E., and Smith, A.G. 2003. Functional analysis of a b-1,3-glucanase gene (Tag 1) with anther-specific RNA and protein accumulation using antisense RNA inhibition. J. Plant Physiol. 160:1367-1373.
- Westberg, C.R., and Smith, A.G. 2003. A novel selection scheme for identifying genes in regulatory pathways. Plant Physiol. Supp. 1121.
- McNeil, K.J. 2003. Analysis of cysteine- and glycine-rich proteins encoded by genes expressed during anther development in tomato (Lycopersicon esculentum Mill.). University of Minnesota, Ph.D. Thesis.
- Carson, T.D. 2003. The application of ISSR PCR for distinguishing creeping bluegrass (Poa annua var. reptans) genotypes. University of Minnesota, M.S. Thesis.
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Progress 01/01/02 to 12/31/02
Outputs
The goal of this project is to understand the molecular genetics of flower development and gene expression. Flower development is a prerequisite for the formation of seeds and fruits of most of the economically important agronomic and horticultural plants. The objectives are to elucidate the function and mechanisms that control flower-specific gene expression, to apply this understanding of the molecular genetics of flower development toward improving horticultural crops, and to train students and post-doctoral researchers. A genetic approach using a conditional lethal gene has been developed to identify genes that regulate flower-specific gene expression. The control region from two anther-specific genes are being used in combination with the conditional lethal gene to select for mutations which regulate anther-specific gene expression. Methanesulfonic acid, ethyl ester mutated populations of plants carrying the conditional lethal gene regulated by the
flower-specific control region have been generated and are being screened for plants that are male-fertile. The fertile plants should carry mutations in genes that are necessary for correct regulation of the anther-specific control regions. To date a single plant has identified as a potential mutant. Preliminary analysis indicates the mutation that results in loss of anther-specific gene expression is recessive to the wildtype allele and may also result in abnormal pollen formation. Experiments to further analyze the mutation and map its location are underway. The function of a gene encoding a glycine-rich protein (GRP) with stamen-specific expression has been characterized. The cellular and subcellular locations of the GRP were determined and the phenotype of transgenic plants with reduced expression levels assessed. The GRP was localized to the outer wall of the pollen, the tapetum (a diploid paternal tissue), and orbicules that line the wall of the pollen sac. This gene product
also impacts the morphology of the outer pollen wall, the viability of the pollen, and its ability to germinate in vitro. Using antisense RNA, the expression of a second GRP gene has been successfully reduced. Unlike the first gene the second GRP gene may be redundant or have little impact on pollen morphology or function. Experiments are underway to introduce reduced expression of both GRPs into a single line of tomato by crossing the two antisense lines. These plants will be analyzed for gene expression and effects on pollen. Male-sterile Petunia lines and male- and female-sterile tobacco lines were produced using stamen-specific expression of the cytotoxic gene barnase. The male-sterility gene caused only a slight morphological change to the anther, but resulted in complete loss of pollen. The female-sterility gene caused a significant reduction in female fertility but not complete female sterility. Alterations in the sterility gene construct were made that are predicted to
decrease female fertility or result in female sterility. The sterility gene was introduced in tobacco and plants will be analyzed for male- and female-sterility.
Impacts
Plant reproduction is an essential step in the development of seeds and fruits and is a major determinant of ornamental value. This research project will increase the understanding of stamen and pistil development, the function of genes in reproduction, and mechanisms that control their expression. This information can be put to direct use in eliminating allergenic pollen production, decreasing nuisance fruit production, increasing flower longevity, increasing the number of flowers produced on a plant, and controlling reproduction and invasiveness. These are very important traits for herbaceous and woody ornamental plants.
Publications
- Rieneke, R., W. Hackett, and A.G. Smith. 2002. Lignification associated with decreased adventitious rooting competence of English Ivy petioles. J. Environ. Hort. 20: 236-239.
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Progress 01/01/01 to 12/31/01
Outputs
The goal of this project is to understand the molecular genetics of flower development and gene expression. Flower development is a prerequisite for the formation of seeds and fruits of most of the economically important agronomic and horticultural plants. The objectives are to elucidate the mechanisms that control flower-specific gene expression, determine the function of genes with flower-specific expression, to apply this understanding of the molecular genetics of flower development toward improving horticultural crops, and to train students and post-doctoral researchers. A genetic approach using a conditional lethal gene has been developed to identify genes that regulate flower-specific gene expression. The control region from two anther-specific genes are being used in combination with the conditional lethal gene to select for mutations that regulate anther-specific gene expression. EMS mutated populations have been generated and are being screened for plants
that are male-fertile. The fertile plants should carry mutations in genes that are necessary for correct regulation of the anther-specific control regions. After confirmation of the mutations through inheritance tests, the regulatory genes will be characterized. The function of a gene encoding a glycine-rich protein (GRP) with stamen-specific expression has been characterized. The cellular and subcellular locations of the GRP was determined and the phenotype of transgenic plants with reduced expression levels assessed. The GRP was localized to the outer wall of the pollen, the tapetum (a diploid paternal tissue), and orbicules that line the wall of the pollen sac. This gene product also impacts the morphology of the outer pollen wall, the viability of the pollen, and its ability to germinate in vitro. Using antisense RNA, the expression of a second GRP gene has been successfully reduced. These plants will be used to determine whether reduced expression results in specific pollen
phenotypes and to develop experiments to test the function of the genes. Male-sterile Petunia lines and male- and female-sterile tobacco lines were produced using stamen-specific expression of the cytotoxic gene barnase. The male-sterility gene caused only a slight morphological change to the anther, but resulted in complete loss of pollen. Under greenhouse conditions, the male-sterility resulted in increased flower longevity in petunia. The female-sterility gene caused a significant reduction in female fertility. The sterile plants are also being used to determine the impact of sterility on the total number of flowers produced by a plant. Other gene constructs are being produced that will cause complete female sterility. In collaboration with Harold Pellet, transformation systems for apple and poplar are in hand and will be used to introduce sterility genes. This will be a first test of the genes for manipulation of fertility in woody perennial plants. Development of efficient cell
culture systems and methods of transformation are underway for other woody perennials continues.
Impacts
Plant reproduction is an essential step in the development of seeds and fruits and is a major determinant of ornamental value. This research project will increase the understanding of stamen and pistil development, the function of specific genes in reproduction, and mechanisms that control their expression. This information can be put to direct use in eliminating allergenic pollen production, decreasing nuisance fruit production, increasing flower longevity, increasing the number of flowers produced on a plant, and controlling reproduction and invasiveness. These are very important traits for herbaceous and woody ornamental plants.
Publications
- Erwin, J.E., A.G. Smith and R. Warner. 2001. Vernalization, Photoperiod and GA3 Interact to Affect Flowering of Japanese Radish (Raphanus sativus L. `Chinese Jumbo Scarlet'). Horticultural Science. Accepted.
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Progress 01/01/00 to 12/31/00
Outputs
The goal of this project is to understand the molecular genetics of flower development and gene expression. Flower development is a prerequisite for the formation of seeds and fruits of most of the economically important agronomic and horticultural plants. Understanding the molecular aspects of flower production may allow the manipulation of these processes to control fertility. The objectives are to elucidate the mechanisms that control flower specific gene expression, determine the function of genes with expression limited to specific tissue of the flower, to apply this understanding of the molecular genetics of flower development toward improving horticultural crops, and to train graduate students and post-doctoral researchers. A genetic approach using a conditional lethal gene has been developed to identify genes that regulate flower-specific gene expression. The conditional lethal gene encodes a hydrolase that converts an inactive pre-herbicide to the active
herbicide. A plant was selected from an M2 population of Arabidopsis that contains the conditional lethal gene but remains fertile when the pre-herbicide is applied. The function of a gene encoding a glycine-rich protein (GRP) with stamen-specific expression is being investigated. The cellular and subcellular locations of the GRP were determined and the phenotype of transgenic plants with reduced expression levels assessed. The GRP was localized to the outer wall of the pollen, the tapetum (a diploid paternal tissue), and orbicules that line the wall of the pollen sac. This gene product also impacts the morphology of the outer pollen wall, the viability of the pollen, and its ability to germinate in vitro. Future experiments are expected to determine how this protein is important to pollen wall function and morphology. Experiments to address similar questions for a second stamen-specific GRP are underway. Male-sterile Petunia lines were produced using stamen-specific expression of the
cytotoxic gene barnase. The male-sterility gene caused only a slight morphological change to the anther but resulted in the loss of pollen. The lines were tested in greenhouse and field trials to determine the impact on flower longevity. When flower longevity was compared between male sterile and fertile plants, there was a significant increase in the life of the flower in the greenhouse. However, when the same comparisons were made in a field setting, the increase of flower longevity in the male-sterile flowers was much less. These experiments demonstrate the promise of male sterility in producing flowers that are longer lived. In collaboration with Harold Pellett, several species of woody perennial plants have been selected that would benefit from male- and female-sterility by decreasing nuisance fruit production, increasing flower longevity, increasing the number of flowers produced on an inflorescence, or controlling reproduction and invasiveness. Work on developing efficient cell
culture systems, methods of transformation, and a model plant for manipulation of reproduction in woody perennials continues.
Impacts
Plant reproduction is an essential step in the development of seeds and fruits and is a major determinant of ornamental value. This research project will increase the understanding of flowering for the manipulation and enhancement of crop production and the development of novel ornamentals.
Publications
- Carson, T.D., D.B. White, and A.G. Smith. 2000. DNA Fingerprinting of Creeping Bluegrass Using InterSimple Sequence Repeats (ISSR). 2000 Annual Meetings Abstracts, American Society of Agronomy, pp 167.
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Progress 01/01/99 to 12/31/99
Outputs
In this project, the function of genes with stamen-specific expression is being investigated. The cellular and subcellular locations of the glycine-rich protein (GRP) 92B was determined and the phenotype of transgenic plants with reduced expression levels assessed. From these and other data, it is hypothesized that this protein is important for maintaining the integrity and morphology of the outer pollen wall. This function also impacts the pollen's ability to effect fertilization and remain viable through dehydration and maturation. Future experiments are expected to determine how this protein is important to pollen wall function and morphology. Experiments to address similar questions for a second stamen-specific GRP are underway. The molecular genetic mechanisms that regulate plant development involve the perception of a signal, the transduction of that signal, the production of specific regulatory proteins, and the activation of transcription. I developed a system
where stamen- and root initial-specific promoters are expressing a conditional lethal gene that allows for direct selection of plants carrying mutations in genes involved in the regulation of stamen and pollen development and the formation of adventitious roots. Several lines have been selected from an M2 population of Arabidopsis that may contain mutations in genes that regulate stamen development. Screening methodologies for adventitious rooting mutants in Nicotiana using the conditional lethal mutant are being tested. The expression of the conditional lethal gene in root initials will also be used to understand the organization and development of these structures. This project uses information and flower-specific promoters to produce cultivars that are sterile. A stamen-specific promoter from a tomato gene that encodes a cysteine-rich protein was used to target the expression of the cytotoxic gene barnase to the stamen. A female-specific promoter from a tobacco gene that encodes a
glucanase was used to target the expression of barnase to the style. Transgenic tobacco plants containing this construct were produced and shown to be completely male sterile and partially female sterile. A second sterility construct was produced that should have resulted in complete female sterility; however, rearrangements of the DNA during transformation inactivated the female sterility portion of the gene construct. Preliminary data from Petunia plants carrying the male-sterility gene show that flowers on these plants last 7-9 days longer than pollinated flowers. In collaboration with Harold Pellet, several species of woody perennial plants have been selected that would benefit from male- and female-sterility due to nuisance fruit production, flower longevity and the number of flowers produced on an inflorescence, or invasiveness. Work on developing efficient transformation systems continues with these plants.
Impacts
Plant reproduction is an essential step in the development of seeds and fruits and is a major factor in determining the esthetics of ornamentals. This research project will increase the understanding of flowering for the manipulation and enhancement of crop production.
Publications
- Fuerstenberg, S. and A.G. Smith. 1999. Molecular characterization of an anther-specific gene from tobacco showing sequence similarity to a tapetum-specific gene from tomato. In press. Sexual Plant Reproduction 12: 250-252.
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Progress 01/01/98 to 12/31/98
Outputs
The project molecular analysis of floral gene expression has the goals of understanding the function of anther-specific genes in the development of pollen, elucidate the mechanisms which regulate anther-specific and developmental gene expression, and to use this information to manipulate plant reproduction. Toward understanding the function of glycine-rich proteins (GRPS) expressed exclusively in the tomato anther it was determined that the 92B GRP accumulates exclusively in the tapetum, the callose wall surrounding the microspore mother cells, and the outer wall of the microspores and pollen. These data suggest that the 92B GRP is an important structural protein of the pollen cell wall. Tomato plants with decreased levels of the 92B GRP had a reduced level of pollen germination and abnormal pollen wall morphology. Normally the outer wall of the pollen cell wall is uniform with stipples at regular intervals. The the outer walls of pollen from plants with reduced
levels of 92B GRP have random invaginations covering the pollen grain. These invaginations are rarely seen in plants with normal levels of 92B GRP. The levels of two other anther-specific GRPs were not altered in these plants. A genetic approach has been taken to elucidate the mechanisms which regulate anther-specific and developmental gene expression. A conditional lethal gene has been fused to two previously characterized anther-specific promoter regions. The gene was introduced into Arabidopsis thaliana, a model organism for genetic studies. The expression of this conditional lethal causes male sterility and therefore no siliques are set on plants containing this gene. A mutagenized population is being screened to identify genes that are required or anther-specific or developmental expression of the transgene. These plants are identified by the production of siliques with seeds. Several plants with full to partial male fertility and therefore containing putative mutations in
regulatory genes have been observed. These plants are being analyzed and there genetic mutations identified. The information we have gathered on anther-specific gene expression and function is being used to regulate the male and female fertility of several plants species. A gene construct has been produced that when introduced into tobacco resulted in complete male sterility and partial female sterility. A second gene construct is being produced that when introduced into plants will result in complete male and female sterility. In order to apply these genes to controlling plant fertility for facilitating plant hybridization, controlling introgression of genes, reducing invasiveness, and increasing flower number and longevity, an efficient gene introduction system must be developed. Tissue culture methods for gene introduction into crabapple, spiraea, and petunia are being optimized. These methods are crucial to the introduction of genes into these plants for controlling fertility.
Plants produced using this approach will have increased efficiency for production of hybrid seed, increased flower longevity and flower number, and decreased ability to become plant pests.
Impacts
(N/A)
Publications
- Reineke, R.A. November 1998. Association between vascular lignin and rooting competence in English ivy. M.S. Thesis, University of Minnesota.
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Progress 01/01/97 to 12/31/97
Outputs
Toward completion of the objective to determine the function of glycine-rich proteins (GRPS) localized in tomato pollen cell walls, antibodies specific to the 92B GRP were used to determine the accumulation pattern and localization of this protein, and antisense RNA was used to decrease 92B GRP accumulation. The 92B GRP accumulates exclusively in the tapetum callose wall surrounding the microspore mother cells and the outer wall of the microspores and pollen. The 92B GRP is developmentally processed from a 12.5 kD protein form into a 9.9, 8.7, and a final from of 7.4 kD that is localized on the mature pollen. Plants with decreased levels of the 92B GRP have reduced fertility with an unusual morphology to the outer surface of the pollen cell wall. These data suggest that the 92B protein is a structural protein for the pollen cell wall and is necessary for full viability of the pollen. Toward completion of the objective to produce male- and female-sterile cultivars of
woody plant species, two crabapple cultivars have been established in culture for in vitro production. These cultivars are being used in experiments to optimize Agrobacterium- mediated DNA transfer. A gene construct designed to ablate the anther and style has been produced and will be used to produce male and female sterile crabapples.
Impacts
(N/A)
Publications
- Lund, S.T., A.G. Smith and W.P. Hackett. 1997. Differential gene expression in response to auxin treatment in the wild type and rac, an adventitious rooting-incompetent mutant of tobacco. Plant Physiol.
- McCune-Zierath, P.D., J.V. Carter and A.G. Smith. 1997. A novel selection strategy for identifying regulatory genes. Plant Physiol.
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Progress 01/01/96 to 12/30/96
Outputs
The goal of this project is to develop a better understanding of the regulation and function of genes expressed during gamete formation. The objectives have been to identify, isolate, and characterize genes that are important to the production of pollen. Effort is focused on 3 groups of genes localized to the tapetal tissue, a single layer of cells surrounding the pollen sac that is essential for the production of pollen. The groups of genes being studied include those encoding glycine-rich proteins (GRP), cysteine-rich proteins, and endo B-1,3-glucanases. Antibodies against a representative GRP have been produced. Accumulation of this GRP occurs only in the anther and is developmentally processed. Localization of the GRP has shown that it occurs mainly in the callose wall of microspores, small vesicles (orbicules) formed in the tapetum, and the outer wall of the mature pollen grain. These results suggest a structural role for this GRP in the microspore and pollen
walls. To determine the function of this GRP, plants with reduced levels of GRP have been produced and are being analyzed. A novel strategy to study genes that regulate anther-specific gene expression is being developed using a gene encoding phosphonate monoester hydrolase that converts glyceryl glyphosate to glyphosate. Plants carrying a stamen-specific promoter driving the hydrolase gene are being used to identify genes that regulate stamen-specific expression.
Impacts
(N/A)
Publications
- DOTSON, S.B., M. LANAHAN, A.G. SMITH AND G. KISHORE. 1996. A phosphonate monoester hydrolase from Burkholderia caryophilli PG2982 is useful as a conditional lethal gene in plants. Plant J. 10 (2).
- MCNEIL, K. AND A.G. SMITH. 1996. Characterization of proteins encoded by tapetal-specific genes in tomato. Plant Physiol. 111: 149.
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Progress 01/01/95 to 12/30/95
Outputs
The goal of this project is to develop a better understanding of gamete development and function. Effort has expanded to include the study of genes localized to the tapetal tissue, a single layer of cells surrounding the pollen sac and genes involved in egg cell development. The tapetal tissue is essential for the production of pollen. The groups of genes being studied include those encoding glycine-rich proteins, cysteine-rich proteins, and endo beta-1,3-glucanases. Based on deduced amino acid sequence and patterns of RNA accumulation the cysteine-rich genes may encode amylase and proteinase inhibitors or lipid transfer proteins and the glycine-rich proteins may provide structure to the pollen or sporophytic tissues. Antibodies to individuals of these groups show that they are localized to the tapetum and the pollen. The function of the reproductive beta-1, 3-glucanase is thought to be the release of microspore and megaspore tetrads. Protein, RNA, and enzyme activity
levels of the Tag-1 gene correlate with release of the microspore tetrads. Inhibiting the expression of this gene using antisense RNA did not cause male sterility. Experiments are under way to determine the extent to which the Tag1 gene contributes to microspore tetrad dissolution. Two clones have been identified that may represent the genes involved in megaspore tetad dissolution. Experiments to determine RNA and protein accumulation patterns for these genes will be used to determine their role in egg cell development.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
The goal of this project is to develop a better understanding of gamete formation in plants. The objectives have been to isolate and characterize genes that are important to the production of pollen. Effort is focused on three groups of genes localized to the tapetal tissue. The tapetal tissue is essential for the production of pollen, however the nature of these required functions is not known. The groups of genes being studied include those encoding glycine-rich proteins, cysteine-rich proteins, and endo B-1,3-glucanases. Based on deduced amino acid sequence and patterns of RNA accumulation, the cysteine-rich genes may encode amylase and proteinase inhibitors or lipid transfer proteins, and the glycine-rich proteins may provide structure to the pollen or sporophytic tissues. The function of the B-1,3-glucanase is thought to be the release of microspore tetrads. Antibodies against a representative of the cysteine-and glycine-rich proteins have been produced using
recombinant proteins produced in E. coli. These antibodies will be used to determine the location of the products in order to help confirm their predicted function and to study the mechanisms that regulate their expression. A strategy to clone other B-1,3-glucanases involved in magaspore tetrad release, microspore mitosis, and pollen germination is being tested. The B-1,3-glucanase genes will be useful in manipulating and determining the function of callose-containing cell walls in plant reproduction.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
The molecular analysis of floral gene expression in tomato is a project to identify, isolate, and characterize genes that are important to the production of pollen. Eight genes have been characterized and have expression localized to the tapetal tissue, a single layer of cells surrounding the pollen sac. The tapetal tissue is essential for the production of pollen, however the nature of these required functions is not known. These tapetal-specific genes are being used to determine the function and the essential nature of the tapetal tissue in development of the male gametes and the mechanisms that control their developmental and cell-specific expression. DNA sequence analysis has revealed that these genes can be categorized as glycine-rich proteins, cysteine-rich proteins, and an endo (beta)-1,3-glucanase. Antibodies are being raised to these gene products to determine their cellular and sub-cellular location. The cysteine rich gene products are similar to several
families of genes including amylase and proteinase inhibitors and lipid transfer proteins. The function of the (beta)-1,3-glucanase is thought to be the release of tetrads (the products of microspore mitosis) to free microspores. Experiments have shown that the Tag 1 RNA is accumulated in sporophytic tissue and not in the gametophyte prior to callose dissolution suggesting post-transcriptional regulation of this enzyme activity. This is the first report of the identification and isolation of a clone representing stamen callase.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
This project focuses on the analysis of genes that are expressed from early meiosis to the dissolution of quartets exclusively in tomato flowers. These genes are low copy in the tomato genome and represent abundant RNAs in the stamen. Seven of the genes have been characterized and have expression localized to the tapetal tissue, a single layer of cells surrounding the pollen sac. Analysis of the deduced amino acid sequence revealed several common characters among these distinct genes. They all contain a hydrophobic amino terminal extension with the properties of a eukaryotic signal sequence. Based on amino acid composition they can be grouped into either cysteine- or glycine-rich proteins. The tapetal tissue is thought to be essential for the production of functional pollen, however, the nature of these required functions is not known. The properties of these proteins suggest that they are cell wall constituents or storage proteins. These tapetal-specific cDNAs will be
used to determine the function and the essential nature of the tapetal tissue in development of the male gametes as well as the mechanisms that control their developmental and cell-specific expression. This information will be used in future experiments to modify the processes leading to pollen formation to control male fertility for hybrid seed production, modify pollen constituents for increased pollen viability or fertility, or expression of these genes in the non-reproductive organs.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
The molecular analysis of floral gene expression in tomato is a project to identify, isolate, and characterize genes that are important to the production of pollen. These genes have several potential uses. The study of these genes will determine the mechanisms that control expression. This information and controlling regions from these genes can be used to target the expression of other genes to the stamen. This targeted gene expression could be used to influence pollen development to produce male sterile plants for hybrid seed production or alter early events in seed or fruit production. The study of the gene products may provide clues on how pollen functions and enable future experiments to alter these functions. This may be useful for changing the interactions of pollen during fertilization or prolonging pollen viability during storage. Eight genes expressed only in stamens from early meiosis to the dissolution of quartets have been isolated from tomato. DNA
sequence analysis of these genes has been used to prodict their function. These predictions are currently being tested. A region from one of the genes has been isolated that controls where, when, and how much product is produced. This controlling region will be used in experiments to manipulate stamen and pollen development.
Impacts
(N/A)
Publications
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Progress 01/01/90 to 12/30/90
Outputs
This research has used tomato flower development to study the processes essential for pollen formation and the molecular mechanisms that control gene expression. The production of functional pollen is a prerequisite to the formation of fruit and seeds and therefore has obvious importance to the plant sciences. The first step toward understanding specific gene functions and the control of gene expression was the isolation of genes that are expressed only in the stamen. The characterization of these genes has included localization of where the gene product is found in the stamen, determination of the DNA sequence of the gene and isolation of DNA sequences that confer stamen-specific expression. Seven of the stamen-specific genes are expressed from meiosis to approximately four days prior to anthesis. The expression is limited to the tapetal tissue which is thought to be essential for pollen development. These clones will be used to determine the nature of the required
functions of the tapetal tissue for pollen development. A region from one of the tapetal-specific genes has been isolated that controls both the cell-specific and developmental regulation. This region can be used to specifically target the expression of other genes to the tomato stamen. Flower-specific gene expression may be useful in modifying the expression of genes isolated from tomato to increase fertilization or cause male sterility for the production of hybrid seed.
Impacts
(N/A)
Publications
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Progress 01/01/89 to 12/30/89
Outputs
Significant progress has been made in the area of physiology & molecular genetics of flower development in tomato. 11 genes selected from an immature stamen cDNA library were determined to be expressed only in tomato stamens. 3 cDNA clones represented genes expressed exclusively in the tapetal tissue of the anther. The tapetal tissue is in intimate contact with the developing pollen & is thought to be essential for the nutrition & proper development of the pollen. It is a key tissue in the reproduction of the plant & is essential for fertilization & fruit set. The other 8, less well-characterized clones, may represent tapetal-specific or gamete-specific genes. We are currently investigating these possibilities. Experiments are underway to use the tapetal-specific clones to learn more about the function of the tapetal tissue & the regulation of gene expression during flowering. Preliminary results indicate that at least part of the DNA sequences controlling
tapetal-specific gene expression have been isolated. These sequences will be used in conjunction with other tapetal-specific regulatory sequences to determine the mechanisms that control the expression of these genes. Other experiments will modulate the level or pattern of gene expression in order to determine the phenotypes of plants that over or under express these genes. These phenotypes can be directly linked to a function for these genes.
Impacts
(N/A)
Publications
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Progress 01/01/87 to 12/30/87
Outputs
Somatic embryoids formed on callus tissue derived from grape floral parts, and in one instance from grape leaves, when the callus was transferred from Murashige and Skoog (MS) medium containing 5 uM 2,4-diclorophenoxyacetic acid (2,4-D) and 1 uM benzyladenine (BA) to MS without 2,4-D and with ir without BA. Embryoids that survived either produced white secondary embryoids, or developed into swollen green structures with cotyledons (germination), or both. Occasionally the germinated embryoids developed into plantlets. During 1987, research was directed at understanding the factors determining embryoid survival, proliferation, germination, and plantlet production. Different levels of salts and sucrose in the medium were associated with differences in the number of new embryoids produced or the germination percentage. Maximum production of secondary embryoids (40% formed at least 5 secondary embryoids) occurred on MS with no plant growth regulators (PGR) and 40 g/l
sucrose. Maximum germination (51%) occurred on MS with 1/2 strength salts and 10 g/l sucrose (germination medium). Various plant growth regulators were tested for their ability to cause germination and/or plant development. Thirty days initial treatment on solid germination medium containing 100 uM gibberellic acid (GA(3)), followed by 60 days on germination medium with no PGR, increased plant production from germinated embryoids from 4.5% in the control group to 13%.
Impacts
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Progress 01/01/86 to 12/30/86
Outputs
Field performance of in vitro propagated blueberry plants (IVP) have far outstripped yield from conventionally propagated plants in three field plantings in different locations in Minnesota. Fruit yields for IVP plants were double those for cutting propagated plants in the first harvest season & the trend continued into second and third harvest seasons. Yield difference is ascribed to greater number of basal and lateral branches. Populus somoclonal variation research: axillary and adventitious shoot cultures of all clones have been increased sufficient for projected future studies. Over 1,000 rooted somoclones produced so far; 500 tested for resistance to Septoria have been field planted for testing for resistance. Adventitious shoots have been obtained from midrib and petiole thin sections in great quantities. Such in vitro thin section culture techniques provide excellent experimental systems for direct observation of morphogenesis. Castanea shoots have been
generated from callus produced following plating of suspension culture aliquots. Similar techniques have resulted in embryogenesis and organogenesis from three grape cultivars. Both Castanea and Vitis systems show great promise for large scale micropropagation systems and somoclonal variation studies.
Impacts
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Progress 01/01/85 to 12/30/85
Outputs
Tissue culture (TC) procedures were developed for in vitro production of callus and buds from shoot tips, needles & cotyledons of white & black spruce (Picea spp.). Blueberry clones 'Northcountry' & MN #367 were proliferated on WPM with 10 ppm 2iP. N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU) treatments caused some shoot proliferation of MN #167 blueberry; other cytokinins were ineffective. Field yields of TC-propagated 'Northblue' nearly 3X that of plants from cuttings (2nd year). Regeneration from hyacinth scale explants decreased after 105 days of source bulb storage at 5C, but did not if stored at 15 or 25C. Heaviest bulblets resulted if source bulbs stored 15C for 70 or 105 days. TC of Castanea dentata and C. mollissima x C. dentata was successful using explants from mature quiescent stems forced in 200 ppm 8-hydroxyquinoline citrate & 2% sucrose solutions. Several other genera responded similarly; both benzyladenine and GA(3) incorporated into tissue via
forcing solution, with subsequent effect on TC. Somaclonal variation studies (with NC For. Exp. Sta.): Populus somaclones produced from several hybrids & variability in response to Septoria observed; studies initiated on Castanea:Cryphonectria interactions. Bacterial contamination reduced after 1 passage on media with antibiotics for azalea TC; but blueberry TC required continuous culture on antibiotic media. Grape callus increased 2-4X when 10M ABA was added to callus medium.
Impacts
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Progress 01/01/83 to 12/30/83
Outputs
In vitro-derived (IVD) plants of 'Northblue' blueberry produced more basal shoots than plants produced by leaf-bud cuttings (CP). IVD plants produced up to triple the CP flower bud no. in field studies. Winter survival in a small scale field study was greater for IVD than CP plants. Cuttings taken from IVD azalea stock plants required less rooting hormone than those from CP stock plants. Explant growth in vitro from IVD stock plants was superior to that of explants from CP stock plants. Alnus in vitro: BA was required for each reculture to assure shoot proliferation and culture survival. 2iP could not be substituted for BA. Developed protocol for in vitro multiplication of Typha glauca (cattail): immature scape c.s. explants on LS medium with 5 mg 2,4-D/L for 10 wks followed by 10 wks LS with 1 mg BA. T. glauca, T. angustifolia and T. latifolia green callus portions recultured with 1 mg BA yielded multiple shoots in 6 wks. Erythronium albidum and E. propullens
proliferated in vitro from lateral or dropper bulb explants on N6 or LS medium. Origin of Leatherleaf Fern organs in vitro discerned; proliferation of Castanea and an aphid resistant Lonicera achieved and meristems cultured from in vitro-produced shoots of dahl were cultured. Successful nodulation achieved on Alnus roots in Salix/Alnus plantings in hydroponics, thus improving Salix growth. IVD azalea plants retarded by daminozide or chlormequat in greenhouse pot studies. 4PUs shown more effective cytokinins than 2iP for in vitro azalea cultures.
Impacts
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Progress 01/01/82 to 12/30/82
Outputs
A tissue culture (TC) medium for azaleas was refined, thus enabling delivery of microstock cultures (MSC) to commercial nurseries to expedite introduction of 4 clones in 1983-85. Low light (30 MuEM - 2sec - 1) applied to MSC enhanced microcutting (MC) production. MC no. increased through 3rd MSC, then declined, but rootability increased through 5th MSC (100). A reduced need for 2ip was denonstrated for repeated MSC. We published first known paper on Alnus TC. In contrast with azalea, Alnus continues to produce numerous MC through 12 or more MSC. Alnus species cultured: A. glutinosa, A. rubra, A. crispa, A. rugosa. Nutrition of Saliz stock plants was linked to no. and rootability of cuttings produced and TC success. High rate of callus formation was obtained using explants from developing Typha scapes (flower stalks) and such calli readily formed roots and occasionally shoots. Mass micropropagation of 'Northblue' blueberries was achieved and MSC were given to
commercial nurseries. In Vitro derived plants produced more crown shoots than plants derived from cuttings. Apparently healthy 'Dark Tiara' dahlias were obtained through meristem culture and re-culture from In Vitro cultures was achieved. Salt tolerant tomatoes grew better on high salt TC media than did conventional cultivars. TC plants of Alnus, Salix, azalea and blueberry have been successfully established in the field.
Impacts
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Progress 01/01/81 to 12/30/81
Outputs
The concept of "microstock" culture (MSC) was successfully explored as a unit for stock plant research. MSC of AZI hardy deciduous azalea (HDA) were given to 3 commercial nurseries for multiplication and possible introduction to the trade. Two more HDA clones were increased to 1000+ via tissue culture (TC). Seven more HDA clones, Syringa, Viburnum, Prunus and Aesculus were brought into TC by direct explanation from field plants. HDA MSC formed more and/or better microshoots (MSH) in TC when cultured under varied light intensities (30 MuEm -2(s) -1 best) or light qualities. Rooting of such MSH was enhanced by these light regimes and pH of 4.0 to 4.5 in rooting medium of peat-vermiculite. GA(3) in MSC medium increased MSH length and handling ease. Successful TC of Alnus glutinosa, A. rubra, A. crispa, 14 Salix clones and lingonberry (Vaccinium vitis-idaea) was accomplished. Stock plant nutrient level was found to influence macropropagation and TC of Salix. Anatomy
of root initiation in Rumohra adiantiformis TC was delineated and 1000+ TC plant established. Correlations between TC of inbred parents and hybrid petunias were established; usually parents with rapid TC ability had progeny of rapid TC ability. Typha TC research was initiated with meristems and developing scapes established as best explant sources for contamination-free TC. A high humidity chamber was refined for direct rooting of MSH. Alnus nodulization and Alnus/Salix interaction studies were begun.
Impacts
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Progress 01/01/80 to 12/30/80
Outputs
Tomato model system was used to illustrate stock plant influence on tissue culture (TC) success: varying benzyladenine (BA) levels enabled predictable morphogenetic responses, roots, callus or shoots; decapitation caused reduced root and biomass production; GA(3) sprays reduced roots produced in TC, but increased callus; BA had no TC effect, but did act as a growth retardant; free-branching mutants ("bushy" and "compact") produced more roots and shoots than apically dominant 'Ailsa Craig' (AC); AC improved in root and shoot production when grafted into "bushy"; "lateral suppressor" mutant produced more roots than isogenic counterpart 'Craigella'. Azalea TC shoots rooted successfully when protocol developed for petunia was used. Promising early results were achieved on Salix and Alnus TC for bioenergy plantations, and on methods for adaptation to soil environment with azalea, Salix and Alnus TC plantlets.
Impacts
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Progress 01/01/79 to 12/30/79
Outputs
Stock plant influence on tissue culture success emphasized. Model systems utilizing in vitro culture of tomato or petunia leaf segments employed to study influence of stock plant nutrition, growth regulator treatments, cultivar, cv-parent interactions, single gene mutants, grafting and mechanical manipulations on subsequent organogenesis. Extremes of N nutrition (high or low) caused reduced shoot prod in tomato, 2000 ppm chlormequat caused increased shoot prod. Shoot no. in petunia varied with cv from 1.5 to over 40/explant. Inbred parents of high shoot producers had 1/10 to 1/4 the shoot no., parents of low shoot prod had few to no shoots. Results from tomato mutant and decapitation studies suggest strong interactions among probable hormone prod sites (leaves, buds, flowers) and in vitro performance. Preliminary experiments with azalea suggest translatability of approach to rooting developed in petunia micro-shoot dipping studies and stock plant and explant
pretreatments.
Impacts
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Progress 01/01/78 to 12/30/78
Outputs
Our research emphasizes two areas: flask atmosphere influence on plant tissues cultured in vitro and stock plant treatments for enhancing tissue culture efficiency. NAA and kinetin increase C(2)H(4) emanation from dahlia leaf explants; KMnO(4) reduction of C(2)H(4) had no effect on dahlia callus, but caused more callus in petunia leaf segments. Close correlation among callus/C(2)H(4)/CO(2) observed. Ethylene injection as low as 0.5Mu1/1 in airtight flasks caused tissue damage, but 5.0Mu1/1 required to cause damage in foil covered flasks. Ethephon in culture medium caused damage, but NAA and kinetin inhibited this damage. Explant pretreatment with 100 ppm AgNO(3) for 1-2 hr caused increased callus, more and longer roots, fewer root hairs, more chlorophyll a and b, higher C(2)H(4) levels. CuSO(4) pretreatment caused lower C(2)H(4) and chlorophyll, but concommittant AgNO(3) reduced CuSO(4) effects. Rhizobitoxine analog explant pretreatments inhibited C(2)H(4) for 2-3
weeks. Eight hr (SD) stock plant and SD incubation caused more callus, C(2)H(4) and CO(2) in dahlia cultures and 2500 ppm daminozide on SD stock plants 1 day before explant removal produced even more callus, C(2)H(4) and CO(2). Stock plant age and leaf position on stock plant influenced performance of tomato leaf explants cultured in vitro.
Impacts
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Progress 01/01/77 to 12/30/77
Outputs
We have devised a system for efficient study of stock plant treatment effects onexplant success in tissue culture. Emphases include: morphology, growth regulators, cultivars, light quality, duration and quantity. In dahlia, short days led to more ethylene and callus production. Short days and 2500 ppm daminozide 1 day before excision caused even more ethylene and callus. Daminozide was ineffective under long days. CCC stock plant treatments may modify GA level resulting in increased shoot numbers from tomato leaf explants. Flask atmosphere: ethylene was reduced by KMnO(4), CoCl(2), a rhizobitoxine analog and AgNO(3), the latter as an explant soak. AgNO(3) soaks of petunia leaf explants also increased root development. Light quality in the stock plant environment has been demonstrated to influence tissue culture success and it is now clear that this response is phytochrome mediated. Red light causes increased stock plant branching and a concommitant increase in
shoot development by petunia leaf explants cultured in vitro.
Impacts
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Progress 01/01/76 to 12/30/76
Outputs
Callus from Dahlia: Callus from basal 1/3 of leaflet > middle 1/3 > tip 1/3. Callus from ray flower segments, greatest to least: tight bud and dark culture, tight bud and light, opening bud and dark--no callus from expanded ray explants regardless of light. Ethylene in Tissue Culture: C(2)H(4) high as 500 ppb foundin dahlia callus culture flasks, associated with presence of fresh leaf or established callus tissue and high (5 ppm) auxin level. C(2)H(4) levels reduced significantly with KMn0(4) crystals placed in flask, but not in medium. Freesiapropagation: Use of aerial corn segments and modified Lindsmaier & Skoog mediumreduced reported time to transplanting by 4-6 weeks. Protoplast Isolation: Isolation of potentially culturable concentrations of Saxifraga sarmentosa 'tricolor' achieved by vacuum infusion with appropriate enzymes. Stock Plant Effects: Established that tissue culture success can be manipulated by treatment of stock plant with cytokinins, varying
light quality.
Impacts
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