Re-examination of the Mendellian and Molecular Genetics of the Arabidopsis thaliana Salk_026354 Line
Mentor 1
Heather Owen
Location
Union Wisconsin Room
Start Date
27-4-2018 1:00 PM
Description
Callose is a plant polysaccharide that surrounds pollen mother cells and separates developing microspores. The gene Callose Synthase 5 (CalS5) of Arabidopsis thaliana is responsible for synthesis of callose, which is associated with proper pollen wall pattern formation. A T-DNA insertion in this gene has been reported to be responsible for plants exhibiting reduced fertility and defective pollen walls. This mutant phenotype requires that both copies of CalS5 contain the T-DNA insertion, meaning the plant cannot carry a copy of “normal” (wild-type) DNA. A previous study has found that, contrary to published literature, polymerase chain reaction (PCR) using gene-specific forward and reverse primers that should not amplify a product in genomic DNA of mutant plants did amplify products. This should not have occurred in mutants with a T-DNA insertion that interrupts the gene; these bands should only be present when a wild-type copy of CalS5 is present. This implies that knockout of the CalS5 gene may not be the cause of the pollen wall defect. To determine whether or not the T-DNA insertion is actually the cause of the defective pollen wall phenotype, flowers from mutant plants have been dissected to confirm pollen wall phenotype under differential interference contrast microscopy and photographed, and DNA has been isolated from these mutants. PCR amplification of mutant DNA using a combination of wild-type and T-DNA primers followed by gel electrophoresis will be performed. If products are formed, the sizes of the amplified products will be determined. If the T-DNA insertion is the cause of the phenotype, the wild-type primers will not show any bands, but a combination of wild-type and T-DNA primers will show a band. Understanding genes that function in pollen development is an important step in manipulating those genes for production of male-sterile lines commonly used in plant breeding.
Re-examination of the Mendellian and Molecular Genetics of the Arabidopsis thaliana Salk_026354 Line
Union Wisconsin Room
Callose is a plant polysaccharide that surrounds pollen mother cells and separates developing microspores. The gene Callose Synthase 5 (CalS5) of Arabidopsis thaliana is responsible for synthesis of callose, which is associated with proper pollen wall pattern formation. A T-DNA insertion in this gene has been reported to be responsible for plants exhibiting reduced fertility and defective pollen walls. This mutant phenotype requires that both copies of CalS5 contain the T-DNA insertion, meaning the plant cannot carry a copy of “normal” (wild-type) DNA. A previous study has found that, contrary to published literature, polymerase chain reaction (PCR) using gene-specific forward and reverse primers that should not amplify a product in genomic DNA of mutant plants did amplify products. This should not have occurred in mutants with a T-DNA insertion that interrupts the gene; these bands should only be present when a wild-type copy of CalS5 is present. This implies that knockout of the CalS5 gene may not be the cause of the pollen wall defect. To determine whether or not the T-DNA insertion is actually the cause of the defective pollen wall phenotype, flowers from mutant plants have been dissected to confirm pollen wall phenotype under differential interference contrast microscopy and photographed, and DNA has been isolated from these mutants. PCR amplification of mutant DNA using a combination of wild-type and T-DNA primers followed by gel electrophoresis will be performed. If products are formed, the sizes of the amplified products will be determined. If the T-DNA insertion is the cause of the phenotype, the wild-type primers will not show any bands, but a combination of wild-type and T-DNA primers will show a band. Understanding genes that function in pollen development is an important step in manipulating those genes for production of male-sterile lines commonly used in plant breeding.
