Progress 09/01/14 to 08/31/19
Outputs
Target Audience:Policy and regulatory agency personnel are the primary audience for this project. As there are only two main mechanisms to introduce genetic variation into crops, mutagenesis and transgenesis, it is imperative to understand the unintended consequences of each mechanism. The results of this project will assess if unintended phenotypic changes occur within soybean seeds more readily in either mutagenized or transgenic populations. The results of the project will likewise be of interest to scientists in the plant biotechnology community, farmers that might be interested in growing genetically modified crops and general public as possible consumers of genetically modified food items. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The primary personal on this project for the duration of this projecthas been a full-time post-doctorate (years 1-3), then a full timemaster level technican (year 3), quarter-time bachelor level technican (year 3) and 3 undergraduate students (years 1-4; extension). All personnel on this project have learned very useful skillsets, includingsoybean transformation and tissue culture techniques, basic genetic principles of growing and screening seeds for homozygous traits. All personnel on the project have also been involved with GFP quantification and protein seed determination. How have the results been disseminated to communities of interest?Presentations have been given at the annual biotechnology risk assessement meeting in Washington DC May each year. Data was also presented as a poster presentation at the biannual soybean molecular biology conference. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: Investigate how the position effect of transgenic insertions influence the seed protein output trait by using a seed-specific ER-targeted and retained GFP expression cassette in transgenic soybean lines (Years 1-4 extension;complete). We have generated all the GFP transgenic lines (50 in total) expressing the ER-retained GFP seed-specific cassette (Years 1-2). Alltransgenic plants have been regenerated to the homozygous stage (Year 3- 4). Analysis showed that GFP expression varied from 0.3-2.5% in the transgenic homozygous seeds compared to 1% GFP/protein (dry weight) in the original parental GFP transgenic line (Year 4). This low variation in GFP amount in the transgenic seeds indicated that an inserted gene expresson is largely determined by the gene expression cassette. We are currently compling the data for a manuscript on total protein and oil content for the transgenic seeds and insertion site of the GFP in each transgenic line. We are completely a high resolution map of the transformation platform cultivar Jack and mapping each of the insertion sites for the transgenic events. The resulting data will encompass the effect of chromosomal position on transgene expression and the effect(s) of transgene expression on collateral gene expression giving rise to unintended altered phenotypes. Further, the results will detail the impact of varying the seed specific expression of GFP on the two dominant seed composition traits in soybean, protein and oil. Objective 2: Investigate the effects of mutagenesis on the production of unintended phenotypes using the transgene encoded expression GFP as a seed storage protein proxy (Years 1-4, extension; complete). A fast neutron collection of mutant seed-specific GFP transgenic seeds were produced (Year 1). Primary screening of the seeds involved protein extraction and determination of percent GFP in over 1,000 seeds (Year 2). Mutant lines that were substantially different from the original 1% GFP/protein (dw) were selected to be regenerated and tested for genome analysis (Year 3). Mutant seeds containing over 2.5% GFP/protein were considered 'high expression' mutants. Likewise, mutant seeds that contained less than the 1% GFP in the parental line were considered 'low expression mutants. The results of the screen were 65 high mutants and 56 low mutants (Year 3). Mutagenized seeds displayed 1.5-15% variation in GFP/protein in their seed content. Eightof the high mutant seeds were analyzed by comparative genomic hybridization analysis using the parental GFP genome to hybridize to each mutagenized GFP event (Year 4). The analysis resulted in determining sections of the genome that were affected by mutagenization and indicate regions in the genome that might result in high protein content in soybean seeds. For example, a homozygous deletion on mutant 185 was detected to span a region encompassing 25 open reading frames (Year 4). We are currently compiling the data of a high resultion genome map of the Jack cultivar used in this mutation study and then aligning transcripts from developing seeds on that map to determine candidate genes that were mutated in each of the high protein mutant lines analyzed (manucript in preparation). This information can be used on how to engineer/breed for high protein seeds in soybean. Current analysis is determining total protein and oil content of the mutant seeds to correlate the variation in GFP expression to the two important output traits in soybean. Data collected in this projectindicates there is much more variation in mutant seeds than in transgenic seeds (manuscript in preparation).
Publications
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Progress 09/01/17 to 08/31/18
Outputs
Target Audience:Policy and regulatory agency personnel are the primary audience for this project. As there are only two main mechanisms to introduce genetic variation into crops, mutagenesis and transgenesis, it is imperative to understand the unintended consequences of each mechanism. The results of this project will assess if unintended phenotypic changes occur within soybean seeds more readily in either mutagenized or transgenic populations. The results of the project will likewise be of interest to scientists in the plant biotechnology community, farmers that might be interested in growing genetically modified crops and general public as possible consumer of genetically modified food items. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The primary personal on this project for Year 4 has been a full-time master level technican, quarter-time bachelor level technican and 3 undergraduate students. The technicans have learned soybean transformation and tissue culture techniques while the undergraduates learn basic genetic principles of growing and screening seeds for homozygous traits. All personnel on the project have also been involved with GFP quantification and protein seed determination. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Transgenesis: Homozygous transgenic seeds will be anayzed for their total protein/oil content and correlate that to the GFP expression levels. Determine by inverse PCR the genome insertion of interesting GFP transgenic events Objective 2: Mutagenesis: Continue genome hybridization analysis of the high mutant events as they will result in candidate genes on how to engineer/breed for high soybean lines. Determine total protein/oil content of mutant seeds, focusing primarily on the high GFP expressing mutant events indentifed in our mutant screen.
Impacts
What was accomplished under these goals?
Objective 1: Investigate how the position effect of transgenic insertions influence the seed protein output trait by using a seed-specific ER-targeted and retained GFP expression cassette in transgenic soybean lines (Years 1-4; 65% complete). We have generated all the GFP transgenic lines (50 in total) expressing the ER-retained GFP seed-specific cassette (Years 1-2). The majority (60%) of the transgenic plants have been regenerated to the homozygous stage (Year 3-4). Analysis showed that GFP expression varied from 0.3-2.5% in the transgenic homozygous seeds compared to 1% GFP/protein (dry weight)in the original parental GFP transgenic line (Year 4). This low variation in GFP amount in the transgenic seeds indicated that an inserted gene expresson is largely determined by the gene expression cassette. Current work is to analyze total protein and oil content for the transgenic seeds and insertion site of the GFP in each transgenic line. The resulting data will encompass the effect of chromosomal position on transgene expression and the effect(s) of transgene expression on collateral gene expression giving rise to unintended altered phenotypes. Further, the results will detail the impact of varying the seed specific expression of GFP on the two dominant seed composition traits in soybean, protein and oil. Objective 2: Investigate the effects of mutagenesis on the production of unintended phenotypes using the transgene encoded expression GFP as a seed storage protein proxy (Years 1-4; 80% complete). A fast neutron collection of mutant seed-specific GFP transgenic seeds were produced (Year 1). Primary screening of the seeds involved protein extraction and determination of percent GFP in over 1,000 seeds (Year 2). Mutant lines that were substantially different from the original 1% GFP/protein (dw) were selected to be regenerated and tested for genome analysis (Year 3). Mutant seeds containing over 2.5% GFP/protein were considered 'high expression' mutants. Likewise, mutant seeds that contained less than the 1% GFP in the parental line were considered 'low expression mutants. The results of the screen were 65 high mutants and 56 low mutants (Year 3). Mutagenized seeds displayed 1.5-15% variation in GFP/protein in their seed content.Five of the high mutant seeds were analyzed by comparative genomic hybridization analysis using the parental GFP genome to hybridize toeach mutagenized GFPevent (Year 4). The analysis resulted in determining sections of the genome that were affected by mutagenization and indicate regions in thegenome that might result in high protein content in soybean seeds. For example, a homozygous deletion on mutant 185 was detected to span a region encompassing 25 open reading frames (Year 4). This information can be usedon how to engineer/breed for high protein seeds in soybean. Current analysis is determining total protein and oil content of the mutant seeds to correlate the variation in GFPexpression to the two important output traits in soybean. Data collected currently indicates there is much more variation in mutant seeds than in transgenic seeds.
Publications
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Progress 09/01/16 to 08/31/17
Outputs
Target Audience:Policy and Regulatory agency personnel are the primary audience for thisproject. As there are only two main mechanisms to introduce genetic variation into crops, mutagenesis and transgenesis, it is imperative to understand the unintended consequences of each mechanism. The results of this project will assess if unintended phenotypic changes occur within soybean seeds more readily in either mutagenized or transgenic populations. The results of the project will likewise be of interest to scientists in the plant biotechnology community, farmers that might be interested in growing genetically modified crops and general public as possible consumer of genetically modified food items. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One post-doctorate associate scientist hasbeen the primary researcher on the project. He is learning mutagenesis, soybean tissue culture and transformation and will soon start learning inverse-PCR methodology to determine genome insertion site analysis. One undergraduate student has helped with the project in the extraction of protein and GFP quantification as well as aid in media preparation for the soybean transformation objective. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Transgeneis.Continue soybean transformation experiments until the proposed total 100 independent GFP lines are produced. Lines need not be homozygous for genome insertion site analysis, so inverse-PCR analysis on the already regenerated 25 GFP lines will be initiated. Objective 2: Mutagenesis: Asthesecond generation of mutant seeds are harvested, analysis of total protein/oil content ofseeds compared to the original mutant GFP line will begin to determine the degree of unintended phenotypesthat occurred in the mutant population thataffected those important agronomic traits.We will initially focus on the highest GFP%/protein mutants as in addition to off-target information they will also offer informationregarding how to engineer a high protein soybean seed. All High mutant plants of interest are currently growing in the greenhouse and DNA will soon be extracted for genome analysis.
Impacts
What was accomplished under these goals?
Objective 1: Investigate how the position effect of transgenic insertions influence the seed protein output trait by using a seed-specific ER-targeted and retained GFP expression cassette in transgenic soybean lines (35% complete). We have used the same GFP construct used to make the original GFP mutagenized line (Objective 2)to generate at least 100 independent lines. Currently, 25transgenic GFPexpressing lines have been regenerated (Year 3). Many transformation experimentsare underway to generate the remaining 75 lines. The 25 regenerated lines are growing in the greenhouse for the next generation and DNA harvested for genome analysis. A total analysis of combined data of insertion site and protein/oil see content will advance the interrelationship of insertion event (genome) with the output trait or phenome advancing the body of information on the position effect in this key US agricultural crop (Year 4). The resulting data will encompass the effect of chromosomal position on transgene expression and the effect(s) of transgene expression on collateral gene expression giving rise to unintended altered phenotypes (Year 4). Further the results will detail the impact of varying the seed specific expression of GFP on the two dominant seed composition traits in soybean, protein and oil. Objective 2: Investigate the effects of mutagenesis on the production of unintended phenotypes using the transgene encoded expression of GFP as a seed storage protein proxy (60% complete). A collection of fast neutron mutant seed-specific GFP transgenic soybean seeds were produced (Year 1). The primary screening of the seeds involved proteinextraction of over 1,000 seeds to analyze for both protein content and GFP percentage of the seed proteome (Year 2). Mutant lines that were substantially different from the original mutagenized GFP line were chosen for growth in the greenhouse and genome analysis. The GFP% determined to be in the original mutagenized line was set at 1% and seeds over 2.5% GFP/protein were considered 'High mutants' and likewise seeds under 1% GFP/protein were considered Low mutants. The screen yielded 156 High mutant lines (with some mutants exhibiting4-10% GFP/protein in seeds) and 104 Low mutant lines with less than 1% GFP/protein (Year 3). Due to a low germination rate of mutants, a total of 121 mutant lines were transferred to the greenhouse and DNA material will be taken for genome analysis. The compiled data of protein/oil content in the mutant seeds displaying altered seed GFP% will advance the interrelationship of mutagenesis with soybean seed output traits (Year 4). Information will be gleaned about the extent mutations effect the two most important output traits in soybean seeds, protein and oil content (Year 4). Additionally, as the screening involves protein content, mutant lines of interest, such as high protein content, will be revealed and analysis of their genome giving insights on how to engineer high protein soybean seeds (Year 4).
Publications
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Progress 09/01/15 to 08/31/16
Outputs
Target Audience:Policy and Regulatory agencies will be interested in the results of this project. As there are only two main mechanisms to introduce genetic variation into crops, mutagenesis and transgenesis, it is imperative to understand the unintended consequences of each mechanism. The results of this project will assess if unintended phenotypic changes occur within soybean seeds more readily in either mutagenized or transgenic populations. The results of the project will likewise be of interest to scientists in the plant biotechnology community, farmers that might be interested in growing genetically modified crops and general public as possible consumer of genetically modified food items. Changes/Problems:The original project proposed producing the mutagenic population of the GFPparental soybean lineby chemical mutagenesis using ethyl methanesulfonate (EMS). We chose tomutagenize theGFP parental seeds by fast neutron mutagenesis as it was safer for the researchers and produces less hazardous chemical waste. What opportunities for training and professional development has the project provided?A post-doctorate associate is the day-to-day researcher on the project. Student workers are also on the project to aid in plant growth, seed collection and seed analysis. The summer of 2016 the post-doctorate was a mentor to a summer high school student performing a short term research project. Students learn the dynamics of growing the important US seed crop soybean and the logistics of performing wet laboratory research. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Production and analysis of 100 GFP transgenic soybean lines. We will continue to generate more independent GFP soybean lines via biolistics of somatic embryos. We will screen each line to ensure they are expressing the inserted GFP cassette before moving lines to the greenhouse.Each line will be grown for a few generations to produce homozygous (all genetically identical) populations.Seeds from each line will be assessed for the amount of GFP/total protein and well as seed composition. Protein and oil are the main constituents of soybean seeds, so both total protein (Bradford assay)and total oil (NMR)will be measured. Protein composition will initially be screened by 2-dimensional gel analysis and for lines showing distinctdifferences acomplete list of protein by mass spectroscopy. Oil compositionwill be determined by GC/MSanalysis. Objectives 2 & 3: Production of 100 mutant lines that differ in GFP amounts compared to parental line and subsequent seed analysis. We will continue to screen the first generation of GFP mutant seeds for seeds that alter in GFP% accumulated in the seeds until at least 100 mutant lines of interest are obtained. Lines will be grown to homozygosity and then in year 4as above will be analyzed for GFP content, overall protein/oil content and protein and oil composition.
Impacts
What was accomplished under these goals?
Objective 1 (year 1 and 2): Transformation of soybean somatic embryos with a seed-specific GFP cassette until 100 expressing lines are produced. Objective 2 (year 1): Determine the optimum conditions to produce a mutagenic soybean seed population using the chemical mutagenic agent EMS. Objective 3 (year 2): Generate a mutagenic soybean seed population and screen for altered GFP seed expression until 100 M1 seeds that have altered GFP accumulation compared to the parental line are identified. The objectives proposed in year 1 involve the production of 100 independent transgenic soybean lines expressing a GFP marker protein and the determination of the optimum mutagenic conditions to produce a 100 GFP seed mutant population. To date we have 12 independent seed specific GFP soybean lines that are moving through tissue culture and have many more transformation efforts underway. We will continue until at least 100 expressing soybean lines have been produced. We are 12% complete for the production of transgenic lines. In years 2 and 3in the proposal,the objective isto regenerate the GFP 100 transgenic lines to homozygosity. The mutagenic objective isto produce 100 mutagenic lines from a single parental GFP soybean line. Lines that have altered GFP protein accumulation in their seeds compared to the parental line will be chosen to be grown to homozygosity and then analyzed for alterations in seed composition traits (protein/oil). To date we have grown the parental GFP soybean line and had the resultant seed mutagenized by fast neutron mutagenesis. Hundreds of mutant seeds were grown in the greenhouse and seed collected. The first generation mutant seeds are currently being screened by measuring both the amount of GFP accumulated and total protein to determine %GFP/ protein in seeds. Seeds that deviate from the parental GFP amount (~3%) are chosen to be grown for another generation in the greenhouse. Currently 16mutant lines have been chosen that displayedup to 5-fold increase (15%) and 2-fold decrease (1.5%) in GFPseed accumulation compared tothe parental GFP transgenic line.Screening will continue until 100 mutant lines that differ in GFP amount accumulated in the seeds have been collected and grown to homozygosity. We are 16%complete inobtaining GFP mutant lines of interest for downstream seed analysis.
Publications
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Progress 09/01/14 to 08/31/15
Outputs
Target Audience:Undergraduate students performing laboratory work for course credit have worked this summer with the post-doctorate on this project. The students learned hands on molecular biology and genetic techniques as well as initiated media preparation, soybean tissue culture and regeneration and transformation. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Post doctorate on the project had the opportunity to attend the two day Soybean Precision Genomics and Mutant Finder workshop held in Minnesota 2015. This workshop will give him hands on knowledge of mutagenesis and will jumpstart his ability to both make a mutant population and subsequently screen for interesting lines. The PD is one of the few academic laboratories that routinely transforms soybean. The post doctorate is learning from the PD on a daily basis how to perform soybean tissue culture and transformation. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period the soybean transformations will continue at a rate of two experiments / month in accordance with the transgenomics objective (a). During this period, the initial transformation experiments will result in regenerated soybean cotyledonary embyro samples that can be tested by both visual fluorescence and molecular techniques if they contain the inserted gene of interest and are expressing the desired trait. Only plants that are correctly expressing the inserted GFP protein will be moved to the greenhouse for further analysis. Seeds for the mutagenesis portion of the project, mutagenesis objective (b),will be in hand within the next few months. When enough seeds havebeen collectedthe first chemical mutagenesisexperiment will be performed.
Impacts
What was accomplished under these goals?
Both transgenic and mutagenic objectives have been initiated. A)Transgenomics objective involves the production of 100 independent expressing seed-specific GFP transgenic soybean lines.The construct to directa seed specific marker gene, green fluorescent protein, expression cassette was made andsoybean transformations initiated.Soybean transformations usingbiolisitcsand embyrogenesishave been initiated and will continue until at least 100 lines of expressing GFP transgenic soybean plants are moved into the greenhouse. B) The mutagenic objective involves making a population of at least 100 mutant soybean lines using an existing GFP stable line as theprogenetor line. The soybean GFP line is being bulked up for seed so chemical mutagenesis can be performed. Currently 50 plants are growing and when seed is collected, chemical mutagenesis will be initiated.
Publications
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