Poplar (Populus spp. and hybrids) are used for pulp, paper and solid wood products. Furthermore, poplar is being developed as a dedicated biomass crop for biofuels and biomaterials. Thus, methods to accelerate genetic improvement to improve woody biomass yield, quality and optimal growth on marginal lands are of considerable interest. One approach is to identify genes that could be manipulated through breeding or biotechnology to achieve these goals. I studied two sets of candidate genes for improving biomass, growth and manipulating flowering time. First, I studied the functions of PopNAC154 and PopNAC156, co-orthologs of Arabidopsis SECONDARY CELL WALL NAC DOMAIN2 (SND2), which are putative regulators of wood cell wall synthesis, the source of lignocellulosic biomass. Second, I studied PopCEN1, PopCEN2, and PopBFT, members of the TERMINAL FLOWER1 (TFL1)/CENTRORADIALIS (CEN) gene family that act as flowering repressors in Arabidopsis and many plants.
I studied INRA 717-1B (P. tremula x P. alba) transgenics with an artificial microRNA (AmiRNA) downregulating PopNAC154 and PopNAC156 (AmiSND2 trees). In a field trial, AmiSND2 trees showed higher mean height and diameter than wild-type (WT). We also observed that AmiSND2 transgenics showed delayed leaf senescence and leaf drop. After conducting controlled environment studies with AmiSND2 trees, I was able to confirm that downregulation of PopNAC154 and PopNAC156 genes does not alter the short daylength-induced bud set and growth cessation but it delays the low temperature induced leaf senescence and leaf drop. Further I was able to show that down regulation of the PopNAC154 and PopNAC156 genes resulted in significantly higher mean plant heights and delayed bud set compared to the WT plants under low soil nutrient conditions. Wood chemistry data analysis of field grown AmiSND2 trees showed that they have a significantly higher cellulose content a lower lignin content compared to that of the WT. Thus, these results show that downregulating the PopNAC154 and PopNAC156 genes has the potential to increase biomass yield and quality.
In a previous study, simultaneous downregulation of PopCEN1 and PopCEN2 genes using RNA interference (RNAi) method caused poplar trees to flower only after two years of growth in the field. I used CRISPR/CAS9 method to knock-out each paralog individually as well as the related gene, PopBFT. The popcen1 mutant trees developed flowers even under in vitro conditions, but popcen2 mutants did not show an obvious phenotype. popbft mutant trees also did not show an obvious phenotype under standard growing conditions. However, when soil nutrient availability was allowed to deplete, the popbft mutants showed lower mean plant height compared to the WT and also showed lower root length and root volume under low Nitrogen conditions in an in vitro assay compared to the WT. These results prove that PopCEN1 gene is directly involved in repressing flowering in poplar and allele-specific mutation should be tested as an approach to accelerate breeding. PopCEN2 and PopBFT might not have a role in regulation of flowering time, and though additional studies are needed, PopBFT appears to have a role in regulating growth in response to nutrient availability. / Master of Science / Poplar (Populus spp. and hybrids) are used for pulp, paper and solid wood products. Furthermore, poplar is being developed as a dedicated biomass crop for biofuels and biomaterials. Genes control the woody biomass yield, and quality among all the other characteristics, thus, methods to accelerate genetic improvement to improve these characteristics is paramount. One approach is to identify genes that could be manipulated through breeding or biotechnology to achieve these goals. I studied two sets of candidate genes for improving biomass, growth and manipulating flowering time. First, I studied the functions of PopNAC154 and PopNAC156, genes which are putative regulators of wood cell wall synthesis, the source of lignocellulosic biomass. Second, I studied PopCEN1, PopCEN2, and PopBFT, genes that act as flowering repressors in Arabidopsis and many plants.
I studied a genetically modified poplar, where PopNAC154 and PopNAC156 genes’ function were reduced. After subjecting these plants to shorter daylength periods and cold temperatures in controlled conditions I was able to show that decreased activity of the PopNAC154 and PopNAC156 genes causes the trees to slowdown the leaf senescence and retain their leaves under low temperature conditions compared to the genetically unaltered wildtype (WT) poplar. I was also able to show that decreased activity of the PopNAC154 and PopNAC156 genes allow the poplar plants to grow better than the WT plants under low soil nutrient conditions.
I used gene editing to switch off PopCEN1, PopCEN2, and PopBFT genes. When PopCEN1 gene was switched off individually the young mutant trees flowered whereas normally trees take six to eight years to flower. However, when the PopCEN2, and PopBFT genes were switched off individually the resulting mutant plants did not show any signs of flowering. Hence, I was able to show that PopCEN1 gene is directly involved in repressing the flowering in poplar plants.
In conclusion, my work identified PopNAC154 and PopNAC156 as candidate genes for manipulation by breeding or biotechnology to increase wood yield, and suggested ways to induce flowering to accelerate breeding through manipulation of PopCEN1.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/101495 |
| Date | 24 June 2019 |
| Creators | Mahendra, Rienzy Ayeshan Rangajeewa |
| Contributors | Forest Resources and Environmental Conservation, Brunner, Amy Marie, Beers, Eric P., Holliday, Jason A., Pilot, Guillaume |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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