Progress 09/01/10 to 08/31/14
Outputs
Target Audience: The target audience is other plant scientists and transgenic biotechnologists, especially those interested in ZFN effectiveness, genetic containment technologies, and tree/forest biotechnology. Changes/Problems: The modifications to project methods and goals were described under results. What opportunities for training and professional development has the project provided? This project has supported training, in part, for two post-doctoral researchers (Amy Klocko and Kelly Vining), and the masters thesis research of one graduate student (Haiwei Lu). How have the results been disseminated to communities of interest? The results have been disseminated via the publications and conference presentations reported in the "Products" section of this and previous reports What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
GENERAL PROJECT OUTCOME The project examined the effectiveness of four zinc finger nuclease (ZFN) pairs that were purchased from Dow AgroSciences. The ZFNs targeted two types of floral genes in poplar. It also examined the effectiveness of negative selection, needed to help produce non-chimeric edited plants in poplar (due to the difficulty of sexual segregation. The overall project result is that the ZFNs had substantially higher cellular toxicity, and substantially lower rates of mutation, than expected. As a consequence we cannot recommend this form of mutagenesis for genome editing or genetic containment in poplar. Fortunately, other types of genome editing methods have been produced are are now being tested for genetic containment in trees in our laboratory and those of others. SPECIFIC OBJECTIVES AND RESULTS Create ZFN pairs and assemble ZFN constructs Collaborating with DOW AgroSciences, we created and selected four ZFN pairs for testing ZFN-induced site-specific mutagenesis in poplar. Two of the ZFN pairs (ZFN800 and ZFN801) target the MADS-box region in both PtAGAMOUS (PtAG) paralogs, and the other two ZFN pairs (ZFN802 and ZFN803) target PtLEAFY (PtLFY). All of the four ZFN pairs were validated in vivo (mice and yeast) by DOW AgroSciences. We assembled the ZFN constructs as originally planned, together with an inducible recombinase for ZFN removal. We transformed one of them (which targets PtLFY) into plants for a month; however, recovery of transgenic plants was poor, likely due to a weak selectable marker provided by collaborator Thompson at USDA. We have therefore abandoned this work until a working marker was provided by Thompson (testing of vectors is in progress; details are explained later). We produced four HSP:ZFN eGFP constructs as substitutions. Each of the four test ZFN pairs was cloned into a vector with a heat-shock inducible promoter (HSP) that drives the expression of ZFN genes. This vector also has a constitutive eGFP reporter to help monitor transformation and chimerism. We also assembled a heat-shock inducible eGFP (HSP:eGFP) construct as a control, which enabled us to study the efficiency of heat shock promoter and estimate ZFN expression level. Test heat shock methods for inducing transgene expression We tested and compared two heat shock methods for their efficiency with HSP:eGFP-transformed explants. In Heat Shock Method 1 (HSM1), we incubated the explants at 42°C for 16 hrs, then shifted them to 22°C to recover for 32 hrs, and incubated them a second time at 42°C for 16 hrs. In Heat Shock Method 2 (HSM2), we incubated the explants at 42°C for 3 hrs every day for 10 days. HSM1 induced a pulse of GFP expression, while HSM2 induced much higher GFP expression. Both of these heat shock methods were used in producing ZFN-transgenic poplars (during early organogenesis stage; i.e., two days after co-cultivation) for either pulse or prolonged nuclease delivery. As discussed below, the long heat shock method resulted in non-regenerable transgenic tissues. Regenerate ZFN-treated poplars with ZFN constructs We performed two rounds of transformations with the HSP:ZFN eGFP constructs and poplar clones INRA 717-1B4 and INRA 353-53. In the 1st round of transformation, we co-cultivated 17,120 explants. However, a much smaller than expected number of regenerated (1,138) and transgenic (259) shoots were produced. All of the transgenic shoots were produced from HSM1 (the "pulse" heat shock method). The reduced transformation rate might be a result of toxicity from elevated ZFN expression. Additionally, we were unable to get any regenerated ZFN802-transgenic events. PCR analysis of the Agrobacterium strain that was used for plant transformation showed an absence of the ZFN transgene, despite multiple efforts to transform it. We suspect this ZFN pair is toxic to the bacteria thus it could not effectively transform plants. The 1st round of transformation test gave an overall shoot regeneration rate of 8.9% (ZFN 802 excluded) and an overall transformation rate of 2.05% (ZFN 802 excluded), both of which were lower than those from the HSP:eGFP construct (32.4% and 5.5%, respectively), suggesting deleterious effects of ZFNs on cell viability. The modest transformation rates with HSP:eGFP also indicate a negative effect of heat shock treatment on plant growth. We performed a 2nd round of transformation to produce more ZFN-transgenic material for analyzing ZFN-induced mutation efficiency. We selectively transformed two of our ZFN constructs (ZFN800 and ZFN803) into plants. We have co-cultivated over 4,500 explants using the two ZFN constructs, and these explants are currently under root induction stage. Based on the GFP expression, we estimate that we will be able to produce about 140 ZFN-transgenic events for each ZFN pair. In the 2nd round of transformation, we used only HSM1 for pulse nuclease delivery during calli induction stage. However, we did not detect any ZFN-induced mutations after analyzing a small sample of 15 transgenic calli (from 7 ZFN800-treated explants and 4 ZFN803-treated explants). We therefore conducted additional heat shock treatments (42°C for 2 hrs per day; every two days; 5 times) during shoot regenerate stage to induce ZFN expression and hopefully obtain desired biallelic mutations. Detect ZFN-induced mutations using high resolution melting (HRM) analysis Using HRM analysis, we screened all of the 259 ZFN-transgenic events (produced in the first round of transformation) for alterations to PtAG1, PtAG2 or PtLFY using locus specific primers. Events showed differentiated melting patterns to wild-types were considered as putative mutants for agarose gel electrophoresis analysis and sequencing. Two putative insertions in PtAG2 were found among the ZFN800-transgenic events in clone 353. This suggests a mutation rate per allele of approximately 0.4% in ZFN800-transgenic poplars. No mutations were found among ZFN801- or ZFN803-transgenic events. These suggest a mutation rate per allele well below 0.4% per explant, much less than anticipated. Further analysis of the putative mutated loci is underway by cloning and sequencing. Test three codA-containing vectors and study the efficiency of negative selection with codA gene We transformed the a vector provided by collaborator Thompson (p409S-nptII::codA) into poplar 717 in late 2011 and could not recover any transgenics; their growth was severely inhibited during the shoot regeneration stage due to a putative problem with its selectable marker identified by Thompson. We received the 2nd vector with the edited selectable marker (p409S-kan2A::codA) in May 2012, unfortunately this construct was not the correct size after transferring into E. coli. In March 2013 we received a new vector from Thompson with a new promoter and modified selectable marker (AtUbi10::codA.nptII), and we have co-cultivated 386 explants in 717. We have confirmed 46 positive events by PCR using codA specific primer and propagated them for further study. Using 9 codA-transgenic plants, we produced 1,299 leaf and stem explants and tested the effect of a variety of concentrations of 5-FC on growth and survival of codA-transgenic poplar tissues in culture (kill curve). We found that 20mg/L of 5-FC did not affect the shoot regeneration of codA-transgenic tissues; surprisingly, 60mg/L of 5-FC promoted shoot regeneration; >200mg/L of 5-FC severely inhibited shoot regeneration. (Our previous results with wild-type plants showed that <500mg/L of 5-FC promoted shoot regeneration, and >750mg/L of 5-FC inhibited shoot regeneration.) These results suggest that the codA-transgenic plants were more sensitive to 5-FC than the wild-type plants. The concentrations of 5-FC between 200 and 500 mg/L could be used for negative selection to distinguish the codA-free tissues from the codA-transgenic tissues.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Lu H, Klocko A, Dow M, Ma C, Amarasinghe V, and Strauss SH. Low Frequency of Heat-induced Zinc Finger Nuclease Mutagenesis of Poplar Floral Genes . American Society of Plant Biologists, Annual Meeting. Portland, OR. 12- 16 July 2014. Poster presentation.
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Progress 09/01/12 to 08/31/13
Outputs
Target Audience: The target audience is other plant scientists and transgenic biotechnologists, especially those interested in ZFN effectiveness, genetic containment technologies, and tree/forest biotechnology. Changes/Problems: It appears from results to date discussed above, that both the rate of transformation and the rate of mutagenesis is far below expectation, thus it is very unlikely that we will obtain any transformants with mutations in both (LEAFY) or all four (AGAMOUS) target alleles. Thus, it is unlikely that we will be able to produce a knock-out mutant that might show a strong sterility phenotype. Unless this situation changes, we will therefore shift our focus to careful characterization of mutation structure, frequency, and off-site mutagenesis, rather than to induce flowering as the heterozygous mutants are not expected to show a sterility phenotype. What opportunities for training and professional development has the project provided? This project has supported work performed by two post-doctoral researchers (Amy Klocko and Kelly Vining), and the thesis research of one graduate student (Haiwei Lu). How have the results been disseminated to communities of interest? The results have been disseminated via the publications and conference presentations reported in the “Products” section of this report. What do you plan to do during the next reporting period to accomplish the goals? We will complete HRM analysis of ZFN mutagenesis and begin cloning and sequencing of HRM identified mutants. In the next year we will focus on characterization of mutation structure, frequency, and off-site mutagenesis.
Impacts
What was accomplished under these goals?
Assembled ZFN constructs Four original planned ZFN constructs—two targeting PtLEAFY (PtLFY) and two targeting PtAGAMOUS (PtAGs) genes—were completed. We transformed one of them (which targets PtLFY) into plants for a month; however, recovery of transgenic plants was poor, likely due to a weak selectable marker provided by collaborator Thompson at USDA. We have therefore abandoned this work until a working marker is provided by Thompson (in progress). Each of the four test ZFN pairs was cloned into a vector with a heat-shock inducible promoter that drives the expression of ZFN genes. This vector also has a constitutive eGFP reporter to help us monitor transformation and chimerism. We successfully cloned all four HSP:ZFN eGFP constructs (two PtAGs-targeting ZFN constructs: ZFN800 and ZFN801, and two PtLFY-targeting ZFN constructs: ZFN802 and ZFN803). We also cloned a heat-shock inducible eGFP (HSP:eGFP) construct as a control, which enabled us to study the efficiency of heat shock promoter and estimate ZFN expression level. Regenerate ZFN-treated poplars with ZFN constructs We transformed all of the four HSP:ZFN eGFP constructs into plants. For each ZFN construct and each poplar clone (INRA 717-1B4 and INRA 353-53), we co-cultivated ~2,000 explants. In total, we produced 1,138 regenerated shoots from ZFN-treated explants. Compared to explants transformed with the HSP:eGFP construct (which gave a shoot regeneration rate of 25%), explants transformed with ZFN constructs showed reduced rates of shoot regeneration: about 10% in both ZFN800- and ZFN801-treated explants, less than 0.2% in ZFN802-treated explants, and about 5% in ZFN803-treated explants. These suggest deleterious effects of ZFNs on cell viability.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Lu, H., A.L. Klocko, M. Dow, C. Ma, V. Amarasinghe and S.H. Strauss. 2013. Zinc Finger Nuclease Based Mutagenesis for Genetic Containment in Poplar. National Institute of Food and Agriculture Biotechnology Risk Assessment Grants Program Annual Project Directors Meeting, June 14, Riverdale, MD. Poster presentation.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2012
Citation:
Strauss, S.H. 2012. Genetic Containment Technology for Poplar Biofuels Plantations. USDA-AFRI, Systems For Advanced Biofuels Production From Woody Biomass In The Pacific Northwest, Annual Meeting, September 10, Boardman, OR. Oral presentation.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2012
Citation:
Klocko, A.L., K.A. Ault, C. Ma, K.J. Vining, J.S. Robertson, M. Dow, H. Lu, E. Elloriaga and S.H. Strauss. 2012. Genetic Containment Technology for Poplar Biofuels Plantations. USDA-AFRI, Systems For Advanced Biofuels Production From Woody Biomass In The Pacific Northwest, Annual Meeting, September 10, Boardman, OR. Poster presentation.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2011
Citation:
Dow, M., E. Etherington, C. Ma and S. Strauss. 2011. Poplar sterility field trial in Corvallis. Tree Biosafety and Genomics Research Cooperative meeting, July 18, Corvallis, OR. Oral presentation.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2012
Citation:
Strauss, S. and K. Ault. 2012. 3rd generation sterility field trial. Tree Biosafety and Genomics Research Cooperative meeting November 15, Corvallis, OR. Oral presentation.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: This project was delayed in starting due to extended negotiations between officials at Dow AgroSciences and Oregon State University over a material transfer agreement to obtain the ZFNs for the study, several months of design, testing, and then delivery of the ZFNs to OSU, and delays in final vector construction due to departure of coPI Tzfira from the project. As a result of the loss of Tzfira, we were forced to produce the constructs at Oregon State University, slowing progress. After many months of work, efforts to produce a recombinase construct that would remove ZFNs with heat induction have been postponed due to problems in cloning of a functional selectable marker by coPI Thompson at USDA. He is still working to produce this construct, but it will be used to only a limited extent in this project, if at all. However, as detailed below, we have now produced a vector with heat-inducible ZFN genes together with GFP to enable monitoring of chimerism, and they are being transformed into plants currently on a large scale. Thus the core project goal of determining the rate of ZFN mutagenesis will be accomplished. The main results this year were: 1. We transferred ZFNs into a vector with a heat-shock inducible promoter that will drive ZFN expression to allow induced mutation to occur during early callus formation. It also has a constitutive eGFP reporter to help us monitor transformation and chimerism. All of the four test ZFNs have been produced and have been transformed into plant tissues. 2. After upgrading our qPCR machine to the StepOnePlus system, we calibrated the HRM system and found that the HRM system is sensitive enough to detect one SNP (i.e. a single base pair change) within a ~1,10bp amplicon. Since ZFN nucleases tend to induce larger base pair changes (i.e. indel mutations up to several tens of base pairs), our HRM targets within PtLFY and HRM analysis system are very promising for detection of nuclease-induced indels. 3. We tested the effect of a variety of concentrations of 5-FC on growth and survival of wild-type and codA-transgenic poplar tissues in culture (kill curve). We found that low concentrations of 5-FC (ranging from 50 to 500mg/L) promoted wild-type shoot regeneration. However, high concentrations (above 750mg/L) severely inhibited wild-type shoot regeneration. 4. We transformed plants with a construct containing the GFP gene to study the chimerism of transgenic-calli over time, and to guide our timing of heat shock. 5. Although we were able to monitor the GFP express over the first three weeks (calli-induction stage), the study was suspended due to the selectable marker problems discussed above. It will be resumed with the new heat-induced GFP construct just created. 6. We transformed our four new heat-shock inducible eGFP/ZFN vector into poplar clones 717 and 353. Currently, we have co-cultivated 2,000 explants for each clone and construct, which should enable us to get about 500 potentially transgenic calli. We were able to identify eGFP expression as early as two days after heat-shock. There were three conference presentations related to containment technologies. PARTICIPANTS: The participants are S. Strauss as PI, J. Thomson at USDA as coPI, C. Ma as transformation technician, Amy Klocko as molecular biologist (constructs), K. Ault as accounts/purchasing manager, and H. Lu as graduate assistant (mutagenesis efficiency). TARGET AUDIENCES: The target audience is other plant scientists and transgenic biotechnologists, especially those interested in ZFN effectiveness, genetic containment technologies, and tree/forest biotechnology. PROJECT MODIFICATIONS: The objectives/approach were modified given the changes in the project that are described under the Outputs section of this report.
Impacts
The project has no outcomes as of yet. The system elements have been studied and the constructs should be produced shortly, after which their effectiveness for mutagenesis will be determined.
Publications
- Vining, K.J., R. Contreras, M. Ranik and S.H. Strauss. 2012. Genetic Methods for Mitigating Invasiveness of Woody Ornamental Plants: Research Needs and Opportunities. HortScience 47:1210-1216.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: This project was delayed in starting due to: 1) more than six months of negotiation between officials at Dow AgroSciences and Oregon State University over a material transfer agreement to obtain the ZFNs for the study; 2) several months of design, testing, and then delivery of the ZFNs to OSU; and 3) delays in final vector construction due to departure of coPI Tzfira from the USA to relocate his laboratory in Israel, and then contractual issues at his University in Israel and with Dow AgroSciences that made it impossible for him to be responsible for producing the constructs. Due to the latter problems, and after discussions with Dow and USDA, we are planning to produce the constructs at Oregon State University in close collaboration with coPI Thompson at USDA. This construct work is expected to begin by the end of November. It is also likely that coPI Tzfira will no longer be able to be a coPI on the project. We have already alerted the program officers at USDA of these problems; once these arrangements are finalized, we will contact USDA for a change in budget allocation and expected removal of Tzfira as coPI. Dow AgroSciences conducted in vivo studies in yeast and mouse cells to determine the effectiveness of more than a dozen ZFN designs targeted at the conserved sections of each of the two target genes, poplar homologs of AGAMOUS and LEAFY. We jointly selected two ZFN pairs for each target gene that showed significant activity in both assays. In preparation for ZFN mutagenesis studies, we have conducted research on development of the system within which the ZFNs will be used this year: A heat shock gene will be used to trigger recombinase expression for removal of the ZFNs after the mutagenesis treatment, thus we compared several promoters and studied the duration of induction needed. Of four promoters analyzed, HSP, 18.2 was the strongest, followed by 81.1, 6871, and 17.5. In stable and transient assays, all four constructs showed GUS leaky expression (i.e., in absence of heat induction), however pCHSP17.5: and 6871::GUSplus showed the lowest leakiness. We tested how duration of heat induction (0,0.5,1,2,4,6,8 and 12 hours) affected GUS expression. The results showed little GUS expression in non-heat treated (control) explants, but a half-hour of heat induction triggered detectable GUS expression. There were few significant differences in expression as a function of duration of heat induction. We compared two candidate constitutive promoters for driving ZFN or reporter gene expression as alternatives to 35S. We found that the 409S promoter showed very strong expression in both transient and stably transformed calli. We also compared two recombinases from the Thompson lab at USDA (CinH and ParA) using a reporter construct for each. CinH gave much stronger GUS expression than ParA in both leaf and stem explants and under both transient and stable expression, and did so at a substantial rate in the absence of heat induction. Its rate was also much higher in leaves than in stem explants. The Ubi7 promoter was also highly expressed both in transient and stably transgenic calli. PARTICIPANTS: The participants are S. Strauss as PI, J. Thomson at USDA at co-PI, T. Tzfira as collaborator (former co-PI), C. Ma as transformation technician, M. Dow as molecular biologist (constructs), K. Ault as accounts/purchasing manager, and H. Lu as graduate assistant (mutagenesis efficiency). V. Shukla at DowAgroSciences is our technical contact and collaborator on ZFN design and delivery. TARGET AUDIENCES: The target audience is other plant scientists and transgenic biotechnologists, especially those interested in ZFN effectiveness, genetic containment technologies, and tree/forest biotechnology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The project has no outcomes as of yet. The system elements have been studied and the constructs should be produced shortly, after which their effectiveness for mutagenesis will be determined.
Publications
- Strauss, S.H. 2011. Why Are Regulatory Requirements a Major Impediment to Genetic Engineering of Horticultural Crops In: B. Mou and R. Scorza, eds. Transgenic Horticulutural Crops: Challenges and Opportunities. Boca Raton, Florida: CRC Press, Taylor & Francis Group.
- Busov, V., S.H. Strauss and G. Pilate. 2010. Transformation as a tool for genetic analysis in Populus. p. 113-133 In: Genetics and Genomics of Populus, S. Jansson et al. eds., Springer.
- Strauss, S.H., D.L. Kershen, J.H. Bouton, T.P. Redick, H. Tan and R. Sedjo. 2010. Far-reaching deleterious impacts of regulations on research and environmental studies of recombinant DNA-modified perennial biofuel crops in the USA. BioScience.
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