Seed germination and stomata wrap up

Posted on April 17, 2024 by Matthew Kaproth

April 16, 2024

By Savanna Newkirk and Lily Flaherty

As the semester progressed, we continued to have numerous opportunities to improve our lab skills and gain more experience. Throughout the last couple of weeks, we have worked on guided experiments and have also created two of our own. In our last blog post, we discussed the inner workings of the beginning of our seed germination experiment. As time has passed, we have had multiple seeds germinate, meaning that we could start planting them. The first to germinate were the controlled spider milkweed (Asclepias viridis), followed by the boiled A. viridis. The wild hyacinth (Camassia scilloides) species was slower to germinate, but there were signs of germination roughly two weeks after the first A. viridis began germinating. Unfortunately, most of our sprouts died within days of being planted. In the coming final weeks of the semester, we will be analyzing our results using statistical methods.

Figure 1. Savanna planting germinated seeds.

Since our last post, we also designed and conducted an experiment that focused on the impact that drought conditions have on stomata. Our experiment involved two species of Oak, Quercus laevis and Quercus virginiana. For each species, we had two plants that we used, meaning four plants total. For each species, one plant was under drought conditions, while the other was watered well. We picked three leaves from each plant (twelve leaves total) and made stomata peels from each. We did this by painting a small strip of nail polish on the underside of each leaf, letting it dry, and gently peeling it off. We then put each peel on a slide and observed them under the microscope. We used the AmScope software to count the stomata and measure the aperture length and width. We measured the length and width of three stomata from each slide made. We have begun to analyze our results. We used a calibration slide to create a scale of pixels to millimeters, then used this scale to convert all of our data from pixels to millimeters. We also found the SPI (Stomatal Pore Index) of each slide that was made. We will continue to analyze our results by using Microsoft Excel or by coding in R.

Figure 2. Stomata from a Q. laevis peel seen through microscope and AmScope software.

We also got the opportunity to conduct a Tetrazolium test (TZ test) on cup plant (Silphium perfoliatum) seeds. A TZ test has the ability to test seeds for viability in a short time frame. To conduct the TZ test, we first acquired three cup plant seeds that had been soaking in water in order to release their hydrogen ions. Following this, we cut the seeds in half lengthwise and placed them cut side down in a petri dish with a TZ solution-soaked filter paper. The seeds were then left to sit at room temperature for 2 days. The TZ solution reacts with the hydrogen ions, staining the live tissue in the seed red while the dead tissue stays white. In all of our seeds we found that the embryo of the seed was stained red, meaning that all three of our seeds were viable.

Figure 3. Viable S. perfoliatum seeds after TZ test.
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RNA Isolations, ABHD2 and DPB Sequencing, and PALD1 PCR

Posted on April 15, 2024 by Rachel Cohen

12 April 2024

By: Paige Hoen, Hailey Ramthun, and Mady Docteur

Figure 1. Mady working on the PCR protocol for the PALD1 samples.

Over the past two weeks, we have continued to research these three genes within the Green Anole Lizards (Anolis carolinensis): PALD1, ABHD2, and DBP. Our goal is to discover the difference in gene expression between the breeding and non-breeding season. So far, we have had more success with our primers for ABHD2 and DPB. For each of these two genes, we identified a primer set that amplifies the correct band size on a gel. With the successful primer sets, we performed a PCR clean-up and sent our samples off for sequencing. Unfortunately, we have been facing difficulties finding a successful primer set for PALD1.

Figure 2. Gel image of RNA Isolation for a liver sample.

After numerous trials and errors with our first three primer sets for PALD1, we concluded it would be best to design new primers. When our new PALD1 primers arrived, we reconstituted them by adding water. Then, we performed PCR using each new primer set. After the PCR was completed, we loaded each sample into a gel and imaged it to see if the correct band sizes for each primer were amplified. Our results from the gel image showed primer dimers for two of the primer sets, and the other primer set showed hardly any bands. This means that we will continue troubleshooting with these primer sets. While working on PALD1 primers, we received our sequencing for DBP and ABHD2. The sequencing showed that our primer sets for both genes are good. Along with working on PALD1 primers, we have spent the past couple of weeks practicing RNA isolation. Eventually, a future step of our project is to perform a qPCR to analyze gene expression. We will need decent RNA isolation samples to complete this. The protocol for RNA isolation is slightly more complex than some of our previous protocols. This is because the RNA in the tissue can degrade if we do not work quickly enough. To practice with this protocol, we began by using liver samples in our first two attempts at RNA isolation. This was to make sure we knew what we were doing before using a brain tissue sample. After doing RNA isolation with liver tissue, we loaded our sample into a gel and imaged it. Both gel images showed that our RNA isolations were successful; showing two dark bands.

Figure 3. Paige and Hailey imaging the gel.

Since we successfully performed RNA isolation on liver tissue, our next step was to perform it again using brain tissue. Brain tissue thaws faster than liver tissue, so it was important that we worked quickly during the protocol. We completed our first brain sample RNA isolation. When we tested our sample on the Nanodrop, we got a concentration of 255.5 ng/µL along with a purity of 2.12. Purity should be close to 2.0, so we were pleased with these results. We ran a gel with the same sample. The gel showed two bands; therefore, we completed the RNA isolation successfully. We did another RNA isolation using brain tissue, and it had good concentration and purity. Next week, we will run a gel using this sample.

Our next steps for the remainder of this semester are to complete two more RNA isolations with a brain sample, ensuring we are consistently successful. Along with this, we will continue troubleshooting PCR for our three PALD1 primer sets. We will try PCR with a dilution, or a temperature gradient, to discover an effective PALD1 primer. An effective primer will amplify the correct band size in a gel electrophoresis with little to no primer dimers. Once we find a primer set that works, we will perform PCR cleanup on it and send it out for sequencing. Overall, we have made a lot of progress these last few weeks in the RISEbio lab. We have found two working primer sets, successfully performed RNA isolations, and are troubleshooting new PALD1 primers. These past weeks have not only taught us new lab skills, but the importance of staying on track, using time effectively in lab, and moving quickly to complete our tasks.

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Drought Experiment with Q. buckleyi

Posted on April 11, 2024 by Matthew Kaproth

9 April 2024

By Ash Waletski and Katelyn Gianni

As the spring semester comes to a close, we have mainly been working on our longest and most detailed experiment so far. Since our last update, we planned and designed an experiment focused on the chronic effects of drought. We chose to work with Quercus buckleyi, the Texas red oak, because it is a close relative to Quercus rubra, the very common Northern red oak that is native to Minnesota (there’s actually a few of these on our campus!). We figured our findings would be more transferable to local ecosystems if we chose an oak species genetically similar to those found nearby.

We want to see if Q. buckleyi saplings that have already acclimated to low water availability, like drought, would have differences in their abilities to uptake water once drought periods are over. We designed our experiment with three groups of five Q. buckleyi saplings each, to control for as many variables as possible (Figure 1). The original states of each group have been maintained for three years in the biology department’s greenhouse, and we changed watering conditions and moved saplings around accordingly about four weeks ago (Figure 2). We had some difficulty moving the saplings that have been grown under well-watered conditions though– they kept getting stuck because of their well-developed roots.

Figure 1. Visual guide for the groups and treatments used.

Every Wednesday since then, we have been looking at the volumetric moisture content left in the saplings’ soil, with the long-term goal of seeing how saplings’ water uptake has been affected. We have been taking and recording percent moisture in each sapling’s soil using a soil moisture sensor (Figure 3). Later this week, we will be diving into another way to see how the saplings have been affected by release from drought– finding specific leaf area, which is the ratio of leaf area to leaf dry mass.

Scientifically, there is a well-documented relationship between a plant’s specific leaf area and the amount of photosynthesis that’s going on inside it; essentially, the denser the leaf, the less photosynthesis it’s able to do. Photosynthesis requires water, so if our experimental group of saplings have different specific leaf areas to either control group, it could be indicative of a change in water uptake. It’s been absolutely fascinating diving into the primary literature behind these processes and learning all about what’s going on inside our little oak saplings. Time and data will tell!

Figure 2. Katelyn rearranging hoses to change watering conditions.

            In the meantime, before we start calculating specific leaf area and between the days that we measure soil moisture, we have been practicing statistical analysis and wrapping up our previous seed germination experiment. Overall, this drought experiment so far has been going quite well and has been a great learning experience. It’s especially been exciting learning new lab techniques. As we mentioned earlier, moving forward our next steps are to calculate specific leaf area, then perform statistical analysis with those values and also with our full soil moisture data. We are very excited to present our future findings to our entire RISEbio team!

Figure 3. Ash measuring soil moisture content using a soil moisture sensor.

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ADAM9 and BHLHE40 PCR Clean-Up and RNA Isolations

Posted on April 8, 2024 by Rachel Cohen

5 April 2024

By: Arya Haala & Elenore Milde

Figure 2. Arya and Elly working on RNA isolations.

Since our last update, we chose two genes to research, ADAM9, and BHLHE40, and designed three primers for each gene. We continued our work with PCRs and gel electrophoresis by adapting them to fit our unique needs. Most notably we worked with a temperature gradient to find the temperature our primers worked best at. As research continued, we threw out two primer sets from each gene, keeping the most effective one for each.

During trouble shooting we used PCR and gel electrophoresis to get clear bands at the correct amplicon size. The size was based on the region of DNA the primers amplify. Once we got the seemingly correct amplicon, we started PCR cleanup to isolate the additives from our gene amplicon. To do this we used 10 μl of our PCR samples and ran a gel to ensure our samples were good. After we confirmed our band sizes were a match, we began our PCR cleanup so we could send out a clear sample for sequencing. We used the remaining ~15 μl of our mixture and 5 volumes of DF Buffer in a column. Centrifuging it and adding 15 μl of Elution Buffer before centrifuging it again. We used the Nanodrop with 1 μl of our solution to measure the DNA concentration and purity of ~1.8. Our purity was a little low, but we continued. We used C1V1=(1 ng/μl)(15 μl) to determine the amount of water needed for sequencing. When this was done, we added the solution, calculated water volume, and 1 μl of the genes forward primer to a PCR tube. This was then sent out for sequencing.

Figure 1. Gel image of temperature gradient

When the samples came back from sequencing, we used the 4Peaks software to analyze our data. Our results came back with a 100% match for ADAM9 and a 95% match for BHLHE40 in the NCBI database. This led us to put the primers away for future use before moving to RNA isolations.

We did two practice RNA isolations on liver tissue to ensure we are ready for real brain tissue. To do this we took one third of a frozen liver sample collected from green anole lizards and homogenized it with 1 ml QIAzol to stop the RNA from degrading. That sat for 5 minutes, then we added 200 μl of chloroform and vortexed. It sat again for 2 minutes before centrifuging at -4°C for 15 minutes. After the 15 minutes was up, we had three layers: clear, white, and red. We pipetted the top clear layer into a new tube getting approximately 600 μl, then added the same amount of 70% ethanol and vortexed to mix. Then we added this new mixture into a column, discarding all flow through. We added 350 μl Buffer RW1 and centrifuged twice then 500 μl Buffer RPE and centrifuged twice more. Following this we used 30 μl of RNA-free water and centrifuged two times using the same water. After this was complete, we used 1 μl of our RNA solution and the Nanodrop to test the concentration and purity.

Both of our liver tissue samples had an ideal purity of ~2 and a concentration that was good for the mass of tissue. Because our samples worked, we used a gel to determine if our RNA had band sizes at 185 and 285 base pairs. Through gel imaging, we determined our samples had good band sizes. Since both of our practice samples went well, we started our first RNA isolation using brain tissue. The difference between brain and liver tissue in RNA isolation is now you will add two steps in the protocol using DNase to make sure all DNA has been transcribed into RNA.

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Be Leaf in Yourself

Posted on March 26, 2024 by Matthew Kaproth

March 26, 2024

By Harper Gillespie & Kayleigh Cottrell

The past month has been consumed with experiments and observations. To start, we planted the seeds that had been germinating into a tray in the greenhouse. We have also begun our own experiment that we recently proposed. Last week, we also conducted a seed viability test to determine if the provided seeds are viable to grow.

Figure 1. Harper adding soil into cells to plant our seeds into.

At the beginning of the semester, we had the opportunity to use seeds and their germination codes to understand their growth. Foxglove beardtongue (Penstemon digitalis) and switchgrass (Panicum virgatum) seeds were chosen and 25 of each were placed into a coffee filter and a Ziploc bag. We used four Ziploc bags, two of which were foxglove and two were switchgrass. From each species, we chose one bag to add mycorrhizal inoculant and water to see if it impacted growth or germination. We hypothesized that the mycorrhizal inoculant would increase the growth of the seeds due to the properties it holds. Mycorrhizal inoculant is used to help the return of mycorrhizal fungi and works well on nutrient-poor soils. After adding water to both, they were placed into the fridge for approximately six weeks. A few weeks ago, the seeds were removed from the fridge and added to the window in a greenhouse bay to enhance their growth. After just a week we noticed sprouts coming from both species. We then added potting soil to a tray of cells and placed the sprouts into them. After continuously doing the process of identifying sprouts and then planting them we only have a few seeds that have not sprouted. We water the seeds and observe their growth twice a week. Presently, the switchgrass has grown the fastest and the tallest, likely due to its germination code (it needs no extra help to grow). The foxglove has been increasingly growing successfully.

Figure 2. Kayleigh observing stomata openings within one of the oak species.

We have also had the opportunity to create our experiment to do for the rest of the semester. We decided to research oak species to determine if they have a memory of prior drought stress. To do so, four oak species were chosen that were from wet (hydric) and dry (xeric) environments. Q. laevis and Q. virginiana have a native range in wet environments while Q. ellipsoidalis and Q. lobata are from dry environments. We will take the oaks out of drought and water them every day instead of every three days. The stomata within each species will be observed. Stomata are microscopic openings found in leaves that are responsible for gas and water exchange and can be closed or opened depending on their environments (Fig. 2). As a result, stomatal peels will be taken before and after the watering treatment is changed. Stomatal peels are taken to observe the stomata in living plants by putting nail polish on a section underneath the leaf and peeling it off. The peels are then put under a microscope for observation. Five stomatal widths on each peel will be averaged from each species. We hypothesize that the oak species will remember prior drought stress. If they remember the prior drought stress, then the stomata of the well-watered oaks will remain closed to hold in their water. The collected stomatal data will be added to a Microsoft Excel sheet. We will then compare and analyze the data to reject or accept our hypothesis.

For the viability experiment, we were provided with cup plant (Silphium perfoliatum) seeds. To test the viability of seeds, we did a Tetrazolium Test (or TZ test). We began by shaving the outer coating off the seed with a razor blade to reveal the inside portion of the seed that we tested. After, the cut seeds were placed in a petri dish on filter paper, and we poured TZ solution over the seeds, so the filter paper was fully saturated. Our petri dish specifically was placed in the fridge because our lab is days apart, to slow the reaction time. The TZ solution will stain the live tissues red, and the dead tissues will have no color change. We will find out soon if the cup plant seeds are still viable.

The growth of foxglove and switchgrass is fascinating to observe. We wonder if the mycorrhizal treatment will show a difference within the next few weeks. In addition, we are excited to begin our experiment and observe the impacts of going from drought to well-watered on stomata.

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