Progress 09/01/16 to 08/31/21
Outputs
Target Audience:Peer scientists; tree breeders; environmental scientists and practitioners; foresters; horticulturists. Changes/Problems:The projecthad delays due to COVID-19-related lockdown. What opportunities for training and professional development has the project provided?A postdoctoral scientist is trained in various molecular and physiological techniques. How have the results been disseminated to communities of interest?Peer reviewed publications have been generated. 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. Generate and select inducible early-flowering male and female poplar lines (Year 1) - 100% completion. This objective was accomplished and a peer-reviewed article already published: "Azeez, A and Busov VB (2019) Improved Heat FT Induction Leads to Earlier and More Prolific Flowering in Poplar. Journal of Botanical Research. 1 (2) 15-17 DOI: https://doi.org/10.30564/jrb.v1i2.1013" Objective 2. Using CRISPR/Cas9 technology, generate strong knock-outs of PopSAP alone and in combination with PopAP2-1/PopAP2-2 in the inducible early-flowering poplar genotypes (Year 2) - 100% completion. This objective was successfully completed. The results from this objective are published in part in the publication summarizing the results of Objective 3 (see below). Objective 3. Study the effects of the single and double CRISPR/Cas9 knock-outs on poplar reproductive and vegetative growth and development (Years 2 and 3) - 100% completion. The objective was completed for the PopSAP knockouts and peer-reviewed article published: "Azeez A, Busov V (2021) CRISPR/Cas9-mediated single and biallelic knockout of poplar STERILE APETALA (PopSAP) leads to complete reproductive sterility. Plant Biotechnology Journal. 19, pp. 23-25 https://doi.org/10.1111/pbi.13451" This study showed that knockout of PopSAP results in complete sterility with no inflorescence initiation. The single and biallelic knockouts in both male and female genotypes caused complete sterility with no initiation of inflorescences. We have also characterized double and triple knockouts (PopSAP and PopAP2-1 and PopAp2-2). The results are showing that contrary to expectation based on the function of AP2 gene in Arabidopsis, the poplar AP2 genes (PopAP2-1 and PopAP2-2) are negative regulators of inflorescence initiation. Knockout of one and particularly the double knockout of PopAP2-1 and 2 leads to significantly increased number of catkins. However, our triple knockouts (popsap/popap2-2/popap2-2) indicate that PopSAP is epistatic to PopAP2 genes in regulation of inflorescence initiation. Knockout of PopSAP in PopAP2 loss-of-function backgrounds as in WT plants leads to complete sterility in both the male and female genotypes. These results indicate that PopSAP is valuable target for engineering robust sterility in poplar. We are working on publishing these results.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Yordanov YS, Ma C, Yordanova E, Meilan R, Strauss SH, Busov VB (2017) BIG LEAF is a regulator of organ size and adventitious root formation in poplar. PLoS One. 2017 Feb 3;12(2):e0171532. doi: 10.1371/journal.pone.0171532
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Azeez, A and Busov VB (2019) Improved Heat FT Induction Leads to Earlier and More Prolific Flowering in Poplar. Journal of Botanical Research. 1 (2) 15-17 DOI: https://doi.org/10.30564/jrb.v1i2.1013
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Azeez A, Busov V (2020) CRISPR/Cas9-mediated single and biallelic knockout of poplar STERILE APETALA (PopSAP) leads to complete reproductive sterility. Plant Biotechnology Journal. doi:10.1111/pbi.13451
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Progress 09/01/19 to 08/31/20
Outputs
Target Audience:Peer scientists; tree breeders; environmental scientists and practitioners; foresters; horticulturists. Changes/Problems:We had delays due to COVID-19-related lockdown. What opportunities for training and professional development has the project provided?A postdoctoral scientist is trained in various molecular and physiological techniques. How have the results been disseminated to communities of interest?Peer reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?We are completing part of the work planned in Objective 3.
Impacts
What was accomplished under these goals?
Objective 1. Generate and select inducible early-flowering male and female poplar lines (Year 1) - 100% completion. This objective was accomplished and a peer-reviewed article already published: "Azeez, A and Busov VB (2019) Improved Heat FT Induction Leads to Earlier and More Prolific Flowering in Poplar. Journal of Botanical Research. 1 (2) 15-17 DOI: https://doi.org/10.30564/jrb.v1i2.1013" Objective 2. Using CRISPR/Cas9 technology, generate strong knock-outs of PopSAP alone and in combination with PopAP2-1/PopAP2-2 in the inducible early-flowering poplar genotypes (Year 2) - 100% completion. This objective was successfully completed. Objective 3. Study the effects of the single and double CRISPR/Cas9 knock-outs on poplar reproductive and vegetative growth and development (Years 2 and 3) - 50% completion. The objective was completed for the PopSAP knockouts and peer-reviewed article published: "Azeez A, Busov V (2020) CRISPR/Cas9-mediated single and biallelic knockout of poplar STERILE APETALA (PopSAP) leads to complete reproductive sterility. Plant Biotechnology Journal. doi:10.1111/pbi.13451" We are working on characterization the double knockouts (PopSAP and PopAP2).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Azeez A, Busov V (2020) CRISPR/Cas9-mediated single and biallelic knockout of poplar STERILE APETALA (PopSAP) leads to complete reproductive sterility. Plant Biotechnology Journal. doi:10.1111/pbi.13451
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Progress 09/01/18 to 08/31/19
Outputs
Target Audience:Peer scientists; tree breeders; environmental scientists and practitioners; foresters; horticulturists. Changes/Problems:A no cost extension was filed due to late start of the project. The delay was due to problems with the visa of the postdoctoral scientist hired on the project. What opportunities for training and professional development has the project provided?A postdoctoral scientist is trained in various molecular and physiological techniques. 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?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. Generate and select inducible early-flowering male and female poplar lines (Year 1) - 100% completion. Poplar male and female clones carrying heat inducible promoter fused to the FT gene were obtained. We first tested the effect of once daily 90 min heat treatment (37oC) on flower induction. We found that flowering was sporadic and took up to 6 weeks for the first flower to appear. We hypothesized that heat-induced expression of FT may be not sufficient to induce early flowering. To mount higher FT expression, we increased the once daily heat treatment to three times daily with 2-hour intervals between the treatments to allow the plants to recover from the heat shock. The repeated induction resulted in almost five-fold increase in FT expression compared to normal once daily heat induction and earlier and uniform flowering in both the male and female clones. We also found much lower levels of induction of the FT transgene in the female clone resulting in production of individual flowers (not catkins) and incomplete flower development. Although the repeated inductive treatments were able to increase the expression of the FT in the female genotype, the levels were still magnitudes lower than the male clone. We will therefore re-transform the female clone with the same construct, regenerate more lines and screen for lines that show better induction and expression of the FT transgene. Objective 2. Using CRISPR/Cas9 technology, generate strong knock-outs of PopSAP alone and in combination with PopAP2-1/PopAP2-2 in the inducible early-flowering poplar genotypes (Year 2) - 75% completion. All four constructs were generated as proposed using multi-target genome editing system. The constructs were sequence-validated, transformed into Agrobacterium were transformed into wild type (WT) and early flowering transgenic poplar clones. For the female clone, we regenerated numerous independent lines for both the WT and early flowering genotype for all constructs. We screened the recovered plants for presence of the transgene and mutations in the targeted loci. We identified lines carrying bi-allelic mutations for the targeted genes with the exception of construct #3, where we were unable to find a bi-allelic mutation in PtaSAP. For this construct we will characterize a PtaSAP heterozygous line, carrying a knock-out in one locus. We have found that Due to inherent genotypic differences in the transformation efficiencies, the transformation for the male clone proceeded at a slower pace. We have now recovered multiple transgenic lines for all constructs and are in the process of bulking up the different lines to allow for initial phenotypic screen and mutation characterization. Objective 3. Study the effects of the single and double CRISPR/Cas9 knock-outs on poplar reproductive and vegetative growth and development (Years 2 and 3) - 10% completion. This objective was initiated for the female clone with knock-out mutations for PtaSAP and PtaAP2_1. The transgenics are being subjected to heat inductive treatment to analyze flower development.
Publications
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Progress 09/01/17 to 08/31/18
Outputs
Target Audience:Peer scientists; tree breeders; environmental scientists and practitioners; foresters; horticulturists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A postdoctoral scientist is trained in various molecular and physiological techniques. 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?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. Generate and select inducible early-flowering male and female poplar lines (Year 1) - 75% completion. Poplar male and female clones carrying heat inducible promoter fused to the FT gene were obtained. We first tested the effect of once daily 90 min heat treatment (37oC) on flower induction. We found that flowering was sporadic and took up to 6 weeks for the first flower to appear. We hypothesized that heat-induced expression of FT may be not sufficient to induce early flowering. To mount higher FT expression, we increased the once daily heat treatment to three times daily with 2-hour intervals between the treatments to allow the plants to recover from the heat shock. The repeated induction resulted in almost five-fold increase in FT expression compared to normal once daily heat induction and earlier and uniform flowering in both the male and female clones. We also found much lower levels of induction of the FT transgene in the female clone resulting in production of individual flowers (not catkins) and incomplete flower development. Although the repeated inductive treatments were able to increase the expression of FT in the female genotype, the levels were still magnitudes lower than the male clone. We will therefore re-transform the female clone with the same construct, regenerate more lines and screen for lines that show better induction and expression of the FT transgene. Objective 2. Using CRISPR/Cas9 technology, generate strong knock-outs of PopSAP alone and in combination with PopAP2-1/PopAP2-2 in the inducible early-flowering poplar genotypes (Year 2)- 50% completion. All four constructs were generated as proposed using multi-target genome editing system. The constructs were sequence-validates, transformed into Agrobacterium and are being transformed into wild type (WT) and early flowering transgenic poplar clones. For the female clone, we have regenerated numerous independent shoots for both the WT and early flowering genotype. These shoots were successfully rooted on selective media. We are screening the recovered plants for presence of the transgene and mutations in the targeted loci. Due to inherent genotypic differences in the transformation efficiencies, the transformation for the male clone is proceeding at a much slower pace. We are in the stages of shoot induction for this clone. Objective 3. Study the effects of the single and double CRISPR/Cas9 knock-outs on poplar reproductive and vegetative growth and development (Years 2 and 3) - 0% completion. This objective will be started as soon as we obtain and validate the transgenic plants carrying CRIPR/Cas9 induced mutations in the target genes.
Publications
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Progress 09/01/16 to 08/31/17
Outputs
Target Audience:Peer scientists;tree breeders; environmental scientists and practioners; foresters; horticulturists Changes/Problems:There was a significant delay in the hiring of the postdoctoral scientist due to long visa processing and approval times. What opportunities for training and professional development has the project provided?Postdoctoral scientist is working on the project and is trained in various techniques. 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?Continue working on the different objectives as planned and described in the proposal.
Impacts
What was accomplished under these goals?
1. Objective 1. Generate and select inducible early-flowering male and female poplar lines (Year 1). Under this objective we will generate transgenic poplars that can flower early and the flowering can be inducible. These genotypes will allow us to test the effects of the genetic modifications of PopSAP as means to impart floral sterility. Trangenic male and female polars expressing FT under heta inducible promoter are being tested at the moment for flower development. 2. Objective 2. Using CRISPR/Cas9 technology, generate strong knock-outs of PopSAP alone and in combination with PopAP2-1/PopAP2-2 in the inducible early-flowering poplar genotypes (Year 2). We will use the newly discovered CRISPR/Cas9 technology for generating strong knock-out mutations in PopSAP and PopAP2. These modifications will be undertaken in the transgenic early flowering plants generated in Objective 1. We expect that the knock-out of PopSAP alone and in combination with PopAP2 will cause sterility with minimum impact on vegetative growth. Constructs were generated as described in the proposal and the various genotypes are being transformed.
Publications
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