Progress 08/01/23 to 07/31/24

Outputs
Target Audience:To date, we have presented results as a poster at our local research day attended by students, educators, and researchers from the region. We have also presented a poster at an international science meeting. PI Klocko gave a seminar talk at Western Colorado University in October 2023 to an audience of professors, graduate students, and undergraduate students. Klocko also presents part of this work to students in BIOL 3830 (Genetics) and BIOL 3230 (Plant Physiology). Changes/Problems:A literally growing challenge for this trial is tree size. The trees are now quite tall, and as clones 717 and 353 tend to form floral buds high in the main crown, these floral buds are not easy to access. Additionally, we are observing canopy closure, meaning crowns of neighboring trees are starting to touch. We are contemplating removal of selected trees to allow for improved access to crowns from trees of high interest. Additionally, floral buds in clone 717 are a bit cryptic, as this clone makes a large vegetative bud on the tip of each branch. We will continue to use our strategy of re-surveying trees for open catkins after the initial survey for dormant buds. We may also use pole pruners or other sampling devices to reach crown buds. What opportunities for training and professional development has the project provided?This project is supporting one graduate student Mallory Nightshade and is the basis for a master thesis project. Mallory has received training in DNA extraction and molecular biology methods, microscopy, field sampling and field photography, and laboratory record keeping. She was able to travel to an international research conference. This project is supporting several undergraduate students. Luis Gilart, Natalie Bondarchuk, Zachary Leach, Hazel Coon and Elenor Harwick, who are assisting the graduate student and receiving training in DNA extraction, leaf morphology measurements, and laboratory record keeping. Natalie Bondarchuk also took part in the field sampling and floral scoring. Undergraduate students Ethan Bertrand and Madeline Keating assisted in field collection of floral data such as photography, floral counts, and surveying floral opening. Two more students, Calvin Brewster and Victoria Conrad, assisted with general trial maintenance, such as weed removal. How have the results been disseminated to communities of interest?To date, we have presented results as a poster at our local research day attended by students, educators, and researchers from the region. We have also presented a poster at an international science meeting. PI Klocko gave a seminar talk at Western Colorado University in October 2023 to an audience of professors, graduate students, and undergraduate students. Klocko also presents part of this work to students in BIOL 3830 (Genetics) and BIOL 3230 (Plant Physiology). What do you plan to do during the next reporting period to accomplish the goals? Floral structure and thus effects on potential for sexual dispersal. Trees will be surveyed again for floral buds and flowers yearly.Trees with sufficient floral buds for collection and indoor flush for microscopy and initial phenotyping.Flowers opening in the field will be scored for timing of floral bud opening and floral form.Male trees will be scored for pollen presence or absence in the field, and female trees for seed presence and seed viability. Vegetative morphology and growth rate, and possible non-target impacts of mutagenesis and ongoing CRISPR expression. Tree size will be measured yearly in the dormant season, and tree viability scored at the start of the growing season.Current leaf data will continue to be analyzed.Trees will be visually surveyed for alterations in crown form within and between trees of the same clonal variety. Stability of phenotypes over years, including whether continued CRISPR expression leads to new mutations or phenotypic changes that impart chimerism within trees. Tree form will be visually assessed yearly along with leaf morphology within and between trees.For heterozygous and WT events, particular attention will be given to floral form across branches of each tree as variable floral form would be indicative of potential target site edits.We will continue with event validation and characterization to have a baseline for comparison for each event.So far no striking phenotype variation has been observed which would indicate presence of chimerism.

Impacts
What was accomplished under these goals? Major goals of the project (the aims) We intend to assess 1. Floral structure and thus effects on potential for sexual dispersal. 2. Vegetative morphology and growth rate, and possible non-target impacts of mutagenesis and ongoing CRISPR expression. 3. Stability of phenotypes over years, including whether continued CRISPR expression leads to new mutations or phenotypic changes that impart chimerism within trees. The main experiment used for all our aims is our field trial. Trees were planted in 2017 (clone 353 and 717) and 2019 (clone 6K10) in randomized blocks by clone with border trees of the same clonal variety. We have 360 total trees representing 43 independent transgenic CRISPR-Cas9 events, 8 Cas9-only control events, and 34 WT control trees. Each event has multiple identical trees (4-8) to provide replication. Aim 1 activities and experiments Data collected In February 2024 we surveyed all trees for the presence or absence of dormant floral buds. These buds are larger and of a slightly different color than vegetative buds. Clones 6k10 and 353 are easy to identify floral buds, clone 717 is more cryptic. For clone 6K10, of the 84 trees, 73 had flower buds. We collected twigs with dormant floral buds for future bud flush analysis. For clone 353 of the 136 total trees, 105 trees had flowers. We collected twigs with dormant floral buds for future bud flush analysis. For clone 717, of the 180 total trees we identified a small number of trees with reachable floral buds (about 5), most were unreachable in the canopy. A resurvey for flowers showed that 27 of the trees in this clone flowered. For all sampled dormant twigs, we completed indoor bud flush (opening) in the lab, photographed the catkins, and checked for either carpels with ovules (clones 6K10 and 717) or anthers with pollen (clone 353). We used a dissecting scope for the assessment of ovules and pollen grains. Events lacking ovules or pollen were noted as being of high interest as these changes indicate loss of female and male fertility, respectively. Floral buds in the field were scored for timing of floral opening, photographed for floral form, and monitored for maturation. For clone 6K10, we collected catkins from 42 trees representing 10 independent transgenic events and 12 control trees. We tested seed viability of 627 seeds and found that 24 were viable. For clone 717, we collected catkins from 19 trees representing 8 independent transgenic events and 2 control trees. We tested seed viability of 380 seeds and found that 29 were viable. As clone 353 is male and does not produce seeds, were did not undertake seed testing for this variety. Summary stats and discussion We again found that two events (DL 345-2 and DL 345-1) in clone 6K10 had small late opening floral buds in both lab and field conditions. These buds lack well-formed carpels and have no ovules and form no seeds. These data show that the female sterility phenotype is stable across floral seasons. All other floral buds from 6K10 trees produced catkins similar to those of control trees. None of the sampled and lab flushed catkins from 717 looked different from WT controls. We identified 3 different independent events targeting AG in male clone 353 (events 167, 193 and 198) that showed green pollen-free catkins when flushed in lab conditions. Examination of two of these events in the field (193 and 198) again showed an absence of pollen grains. Control trees and an event without target site changes had abundant pollen. These data are indicative of AG targeting leading to male sterility. As this is a very small sample size, we did not perform statistical analysis on the floral fertility data. Aim 2 activities and experiments In the dormant season we completed a set of tree size measurements (height and diameter) that will be used to quantify tree growth and overall performance. In spring 2024 we assessed the survival of all trees by checking for leaf flush (leaf opening). All trees from clone 353 were alive in early 2024, one tree from clone 717 was scored as dead, as were two trees from clone 6K10. Overall survival is quite high. As we currently have 3 years of leaves collected, scanned, and measured, we did not undertake a leaf collection in 2024. We did survey canopies for overall leaf form. Summary stats and discussion No highly variable leaf forms were observed within individual trees in the 2024 season to date. We have completed all leaf trait measurements and checked data for outliers. Leaf size, leaf area, and petiole dimensions appear to be normally distributed. ANOVAs for our 6K10 data show that control trees, sterile trees, and edited but non-sterile trees all have similar overall vegetative traits. We did observe a potential difference in leaf patterning at branch ends for our two sterile events, where it appears the trees have most leaves clustered at the branch tip rather than distributed along the length. We are investigating ways to score and quantify these observations. We also found that our floral sterility phenotype for our two LFY events in clone 6K10 is stable across flowering seasons. These two events make reduced catkins lacking ovules or seeds. Aim 3 activities and experiments Data collected We collected branch samples and leaves for DNA extraction and analysis from all events. We are re-checking target site alleles in clone 6K10. We are validating the presence of the Cas9 transgene in our CRISPR-Cas9 events and Cas9-only control events. For this process, we optimized a DNA extraction protocol, ordered and obtained primers, extracted DNA, and used PCR to check for Cas9. Summary stats and discussion We have validated the presence of the Cas9 transgene in 6K10 and 717 events and Cas9-only controls. We have primers that successfully amplify bands for our target genes in clone 6K10, these are being used for target site analysis. Data validation consists of band presence or absence (Cas9), along with band size (target genes LFY, AG1, AG2). WT trees that lack transgenes are our negative control for Cas9 and the positive control for target genes along with representing the size and sequence of non-edited target genes. We are in the process of cloning alleles and sequencing target sites. Initial PCR amplification confirmed that some events have a large deletion in the LFY target gene. Events with WT phenotypes all have a target site band of the same size as WT trees. We will use DNA sequencing to confirm specific allele sequences and will compare them to WT.

Publications

Progress 08/01/22 to 07/31/23

Outputs
Target Audience:Our target audience consists of other professors, researchers, graduate and undergraduate students, regulators, and the general public. This audience will be reached through presentations at international scientific meetings, local conferences, poster presentations, scientific manuscripts, and talks in classrooms. Changes/Problems:A main interest in this work is floral form and floral fertility. Our February 2023 floral bud survey on all trees identified no floral buds on the male 353 or female 717 trees. Re-surveying in later spring when poplar flowers emerge (flowers are larger than floral buds, and open before the leaves) again showed no evidence of flowers on these two groups of trees. Our efforts are currently focused on molecular characterization of trees as well as vegetative performance, and we have leaves collected from all experimental trees for assessment of target genes and leaf traits. We have expanded our experiment to include a set of early-flowering Populus alba female clone 6K10 trees that were planted in 2019 at this same field site and were generated using the same CRISPR-Cas9 vectors as the 353 and 717 trees. These trees did show flowering in spring 2023. One event from these trees had very small late-opening floral buds in the lab. Field evaluation failed to identify these floral buds, perhaps due to small size, late opening, and bud scarcity (4 or fewer per tree). In future years the individual branches will be flagged to facilitate ease of floral bud monitoring. What opportunities for training and professional development has the project provided?Opportunities for professional training This project is supporting one graduate student Mallory Nightshade and is the basis for a master thesis project. Mallory has received training in DNA extraction and molecular biology methods, microscopy, field sampling and field photography, and laboratory record keeping. This project is supporting several undergraduate students. Luis Gilart, who is assisting the graduate student and receiving training in DNA extraction, leaf morphology measurements, and laboratory record keeping. Undergraduate students Ethan Bertrand and Madeline Keating assisted in field collection of floral data such as photography, floral counts, and surveying floral opening. Two more students, Calvin Brewster and Victoria Conrad, assisted with general trial maintenance, such as weed removal. 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?Plan for next reporting period for goals not yet completed Floral structure and thus effects on potential for sexual dispersal. Trees will be surveyed again for floral buds and flowers yearly.Trees with sufficient floral buds for collection and indoor flush for microscopy and initial phenotyping.Flowers opening in the field will be scored for timing of floral bud opening and floral form.Male trees will be scored for pollen presence or absence in the field, and female trees for seed presence and seed viability. Vegetative morphology and growth rate, and possible non-target impacts of mutagenesis and ongoing CRISPR expression. Tree size will be measured yearly in the dormant season, and tree viability scored at the start of the growing season.Leaves will be collected each growing season for trait analysis.Trees will be visually surveyed for alterations in crown form within and between trees of the same clonal variety. Stability of phenotypes over years, including whether continued CRISPR expression leads to new mutations or phenotypic changes that impart chimerism within trees. Tree form will be visually assessed yearly along with leaf morphology within and between trees.For heterozygous and WT events, particular attention will be given to floral form across branches of each tree as variable floral form would be indicative of potential target site edits.We will continue with event validation and characterization to have a baseline for comparison for each event.

Impacts
What was accomplished under these goals? Major goals of the project (the aims) We intend to assess 1. Floral structure and thus effects on potential for sexual dispersal. 2. Vegetative morphology and growth rate, and possible non-target impacts of mutagenesis and ongoing CRISPR expression. 3. Stability of phenotypes over years, including whether continued CRISPR expression leads to new mutations or phenotypic changes that impart chimerism within trees. The main experiment used for all our aims is our field trial. Trees were planted in 2017 (clone 353 and 717) and 2019 (clone 6K10) in randomized blocks by clone with border trees of the same clonal variety. We have 360 total trees representing 43 independent transgenic CRISPR-Cas9 events, 8 Cas9-only control events, and 34 WT control trees. Each event has multiple identical trees (4-8) to provide replication. Aim 1 activities and experiments Data collected In February 2023 we surveyed all trees for the presence or absence of dormant floral buds. These buds are larger and of a slightly different color than vegetative buds. Of our 96 total trees in clone 353 none had floral buds. Of our 180 total trees in clone 717 none had floral buds. Of the 84 trees in clone 6K10, 19 had floral buds, representing 4 controls and 8 events. We collected twigs with dormant buds from 9 of these trees representing 6 of 8 events and 3 controls for indoor bud flush (bud opening). We completed bud flush on all collected samples, photographed the buds, and used a dissecting microscope to examine floral form. Floral buds in the field were scored for timing of floral opening, photographed for floral form, and monitored for maturation. Mature catkins (seed pods) were collected from 14 trees representing 7 independent transgenic events and 3 control trees. We tested seed viability of 20 seeds per tree, and found none of the seeds were viability. Summary stats and discussion We found that two trees in event DL345-2 had smaller floral buds than WT trees. We collected buds from one tree for indoor bud flush and found that the catkins from this tree opened slowly and lacked carpel or ovule development. The buds from the two other LFY-targeting trees and one AG-targeting tree flushed in lab conditions were comparable to WT and ovules were observed in these samples. As this is a very small sample size we did not perform statistical analysis. Aim 2 activities and experiments Data collected In the dormant season we completed a set of tree size measurements (height and diameter) that will be used to quantify tree growth and overall performance. In spring we assessed the survival of all trees by checking for leaf flush (leaf opening). All trees were alive in spring 2023. In May 2023 we collected 3 leaves from each tree for vegetative performance. Leaf traits include total chlorophyll content, leaf area, leaf dry weight, blade length, blade width, petiole length, and petiole width. The chlorophyll, area, and weight measurements are complete, length and width measurements are ongoing. During the leaf collection, trees were visually surveyed for variability in leaf and tree form within individual trees and between trees of the same clonal type. Summary stats and discussion No highly variable leaf forms were observed within individual trees in the 2023 season to date. We are in the process of completing all leaf size measurements (blade and petiole dimensions). Once all leaf trait data is complete for the 2023 growing season, we will use statistical analysis to determine if any constructs, gene targets, or events are significantly different from control trees of the same clonal variety. We will check data for outliers and normality. For data with a normal distribution, we will perform t-tests and ANOVAs. For non-normal distribution, we will either transform data (log transformation), or use other methods such as Chi-squared analysis. In future years we will compare data across growing seasons. These data will be particularly important for overall tree growth and performance. Aim 3 activities and experiments Data collected We collected branch samples and leaves for DNA extraction and analysis from all events. We are validating the presence of the Cas9 transgene in our CRISPR-Cas9 events and Cas9-only control events. For this process, we optimized a DNA extraction protocol, ordered and obtained primers, and have extracted DNA from trees representing all events. Summary stats and discussion We have validated the presence of the Cas9 transgene in 6K10 and 717 events and Cas9-only controls. We have primers that successfully amplify bands for our target gene, these will be used for target site analysis. Data validation consists of band presence or absence (Cas9), along with band size (target genes LFY, AG1, AG2). WT trees that lack transgenes are our negative control for Cas9 and the positive control for target genes along with representing the size and sequence of non-edited target genes. ?

Publications