Jessica Oswald
From Shiu Lab
Welcome! My project in the Shiu Lab is to determine the reasons in terms of fitness advantages why within Arabidopsis thaliana there are both tetraploid and diploid ecotypes. Questions include:
What are the fitness/phenotypic differences between polyploid and diploid ecotypes?
What are the phenotypic responses to different abiotic and biotic stresses by these ecotypes?
What generalizations can we make about tetraploids?
Answering questions like these may give us some insight about how plants and other organisms evolve after whole genome duplications.
Some things about me: I enjoy birding, hockey, anything outdoors, and from my recent experiences I enjoy research very much. I have an Associate's Degrees from Kellogg Community College in science and art and a Bachelor's Degree from Michigan State University in zoology. I am currently applying to graduate schools and hoping to study topics dealing with avian species, conservation, and climate change.
Contents |
January
The purpose of Dr Lehti-Shiu's and my research project is to compare polyploidy ecotypes of Arabidopsis thaliana, especially tetraploids (4n), to diploid (2n) individuals. We want to examine the differences in fitness levels and phenotypes of Warschau and M7943s with many other diploid ecotypes. I have begun planting the various ecotypes and the few different tetraploids. We initially wanted to compare these tetraploids with their parental ecotypes but have not been able to find them with any certainty. We also wanted to confirm that the ecotypes we chose were indeed tetraploid so we examined their cytometry graphs. Dr Lehti-Shiu and I have begun thinking about which characters to compare in our plants. So far we have determined that bolting time, plant diameter, root growth, and leaf numbers would be a good starting point. We also want to compare the Swedish ecotypes and see if perhaps the tetraploid ecotype we have planted can grow properly without going through a vernalization period. After these initial examinations of fitness and phenotype we want to see how the tetraploids and diploid ecotypes respond to different abiotic and biotic stresses. These past few weeks I have learned many new techniques. These include sterilizing seeds and making plant media. I have also learned to how to do DNA preps and have successfully ran two gels. Since then, I have planted my germinated seeds and am currently attempting to make an experiment log to take and organize my notes. I am very excited to start observing our plants.
February
This month I observed my plants that I sterilized, plated, and planted in January. Superficially, it appears that Warschau and one its closer relatives, Landsberg, have similar phenotypes. Also this month I have planted my vernalized Swedish ecotypes along with the known Swedish tetraploids M7943s (vernalized this time) and CS 6186. I will begin observing them in the following couple of weeks. We have also this past month ordered more closely related ecotypes to Waschau, which will be very exciting to work with and compare.
I have also been observing Dr Lehti-Shiu’s work and I have helped with some preparations for experiments. I have grown overnight bacterial cultures, performed a PCR and ran a gel with the products, and made LB. I have also observed Arabidopsis being transformed by agrobacterium. I have also helped plate Arabidopsis seeds on various media to see any phenotypic responses. Unfortunately, we had some contamination and may have to redo this experiment.
I have also been preparing for the Midwestern Section Annual Meeting that I will be attending on the weekend of March 24. I will present my work that I have done so far along with any findings. I have written my abstract and now I have to get to work on my poster for my current project.
March
During the month of March I spent most of my time in the lab preparing for the ASPB meeting during the weekend of March 25. I wrote my abstract during the first week in March and then made my poster. My findings for this first group of diploid individuals (Ler, Rsch-2, Col, Per-1, Tsar) compared with Warschau, the tetraploid, were that there wasn’t an obvious phenotypic difference this is what I presented in my poster. These comparisons only took into account, rosette diameter and flowering time based on leaf number, bolt time, and flower time. I will perform stress experiments and also continue to take measurements on the plants. There should be some type of difference between the groups in my opinion; I just haven’t found it yet. This month I have also plated the diploid ecotypes that are closely related to Warschau; they are currently germinating in the growth chamber. I have also been taking measurements on the Swedish diploid and tetraploid ecotypes that I planted in February. So far I have superficially observed a slower bolting time, a smaller diameter, and leaf number in the tetraploids. There are also differences in the overall color and texture of the leaves. I am still waiting to take accurate measurements of the tetraploids because they haven’t bolted yet.
April
This month I have plated more Swedish ecotypes to compare to the two tetraploids from this region, which includes M7943s and M3385s. I had to repeat this experiment because these tetraploids failed to bolt during the month of March. I am, also, in the process of repeating the Warschau comparison with its closest relatives. I plated some seeds in order for Melissa and Emily to perform flow cytometry. From my first comparison (Warschau and random ecotypes) I am collecting seed and taking bolt height measurements.
May
This month I have been monitoring my Warschau (wa-1) relatives that I plated on April 19 and planted on May 2. As of today the wa-1 ecotype resembles closely landsberg (ler) and landsberg erecta. I am looking at features such as rosette diameter, bolting time, flowering, and flower diameter. These are the same features I examined before in my earlier experiments the only addition was flower diameter because according to Aneuploidy and Genetic Variation in the Arabidopsis thaliana Triploid Response by Henry et al. in 2005 flower size increases with ploidy levels. So far only ler, ler iso, and wa-1 have bolted and flowered and it appears that they all have approximately the same flower diameter. The other seven ecotypes have not bolted yet and since these three have approximately the same flowering time and rosette diameter they are the most similar. The only conclusion so far that I can make about my results would be that wa-1 is a tiny plant with early flowering time but does not differ from ler. Perhaps all three of these ecotypes have a fitness advantage to their later flowering time but bigger relatives. It will be hard to make this conclusion with out stress experiments which I have begun to perform. I have noticed from collecting seeds from my previous experiment comparing wa-1 that the tetraploid seeds are plumper and larger than diploid seeds, this has already been found also by the Henry et al. (2005) in the above paper.
The Swedish ecotypes, except for a couple of Stockholm, that I have been monitoring since April 26 failed to bolt I assume because I did not vernalize them for long enough which was for two weeks. The tetraploids from February have also failed to bolt. In attempt to determine how long the tetraploids, Skulesburg, and Heneriksfal need to be vernalized I have plated the seeds again. This time I have place them in the refrigerator and will leave them there for 30 days or 60 days and then plant them after those time periods. Placing the grown plants in a cold room is an option but my plants are heavily infested with fungus gnats and due to the risk of infesting everyone else’s plants I have chosen not to. The evidence suggests that the tetraploids require a much longer vernalization period than the diploid Stockholm. Since a tetraploid has double the gnomic material does this mean that the vernalization period is twice as long? Also what genes are responsible for this lack of inactivity? What role does the gene Frigida play? I am also interested in what this means in an ecological sense. Would the tetraploid have higher fitness in colder winters, perhaps farther north, or does this protect the plant against freeze/thaw/freeze periods towards the end of winter? Cold experiments are necessary to answer some of these questions.
This month I have also begun the stress experiments I have been planning. I have started with salt stress to see if perhaps the tetraploids have an advantage. I have used both Swedish, Warschau relative ecotypes, and the three tetraploids. So far I have not observed the tetraploids having a higher fitness in high salt environments. I plan on measuring root length, number of lateral roots, perhaps number of leaves, and general survivability. I am performing these experiments on plates placed vertically in the growth chamber. I concerned about this experiment because my plates have accumulated a lot of water and contamination from mold looks like it will be a problem. If so I will at least be able to determine survivability. I also want to look at wounding stress, competition, osmotic, drought, cold, and heat stress. I will continue these when there is available space as well as a reduction in the number of fungus gnats.
June
This month I started my stress experiments on plates. I added mannitol and salt in different concentrations and together in regular ms plus sucrose plates. So far I have been having a hard time with contamination and also they are slow to start growing on this media. Both of these problems together have caused me to have to redo these experiments through out this month.
One stress experiment that I managed to get data from was the salt stress plates from May. I measured root length, number of viable seeds, cotyledons (condition/number visible), and I recorded plant color. Ecotypes Columbia and Stockholm appeared to perform the best under the conditions and Columbia had the longest roots and on average its 2 cotyledons were green, yet it did not have the highest percentage of viable seeds. Compare this to the least viable seeds of Ler, the shortest roots CS3114, and RSCH-2 had an average of one cotyledon grown. The ecotypes all had differences in one area of fitness or another and no ecotype was the worst overall. I would like to repeat this experiment and attempt to plant the seedlings when large enough and contamination level permitting. Apparently vertically growing seeds that are on plates creates an environment that mold easily grows in (probably from all the standing water).
After discussing the tetraploid ecotype Warsaw (Warschau) with the other members in the lab, it was all agreed upon that it will be difficult to determine if a fitness difference is the product of ploidy level or of diversification. This is because Warsaw does not share its habitat with the closest relative, Ler. So, I am going to focus on the tetraploids and the ecotypes from Sweden because they all live in the same region. This way it could be easier to phenotypically categorize differences. Also, Warsaw does not appear to be the classical tetraploid. It does not have delayed flowering (it flowers early), thicker stems/leaves, or large flowers (something that I have been measuring) these polyploid characteristics are all according to the paper by Ramsey and Schemske (2002).
This being said the Swedish ecotypes that I placed in the refrigerator on May 29 for a 30 day vernalization period were placed in the growth chamber on June 27. The seedlings grew quickly and I planted them on July 2. I will compare these ecotypes and hopefully they will bolt and flower within a reasonable amount of time and together. I am going to place these under continuous light. Also I would like to begin to perform more stress experiments with these ecotypes.
Ramsey, Justin and Douglas W. Schemske. “Neopolyploidy in Flowering Plants.”
Annual Review of Ecology and Systematics, 33 (2002): 589-639.
July
This month I have been observing ecotypes from Sweden (CS3114 (4N), CS6183 (4N), CS1535 (2N), Henriksfjall (2N), and Skuleberget (2N)). I am primarily concerned with there bolting and flowering time, rosette diameter, leaf and flower bud number, and flower width. These plants underwent a 30 day ‘vernalization’ in the refrigerator and once 30 days was reached I planted them and have continued growing them in continuous light. I growing them under this condition in order to entice the ecotypes to bolt and flower earlier or at least at similar times to each other. Previously, I have found that the tetraploids within these Swedish ecotypes took about 130 days to bolt and flower compared to the ecotype Stockholm (2N) that takes around 27 days once planted. These ecotypes under continuous light appear to be healthy and bolting and flowering quickly. So far the tetraploids (CS 3114 and CS 6183) have not bolted but I did not expect them to be early flowering.
I have been continuing to take data on Warschau (wa-1) and its ‘relatives’ ecotypes. Wa-1 and Landsberg are currently drying down and I’ll start to weight the seeds that they produced soon. There are many ecotypes including CS994, CS 970, and CS 6044 that just now are bolting and flowering; while CS 22592 which is more closely related to Wa-1 than many of the others hasn’t even bolted yet. This again reaffirms that Wa-1 and Landsberg are phenotypically very similar and perhaps they are very different and far removed evolutionarily from their relative ecotypes. These ecotypes have been under some stress in the growth chamber from powdery mildew and a fungus gnat infestation. I have been having a rather difficult time counting the number of leaves of the ecotypes of CS994, CS 970, and CS 6044 because they have a lot aerial rosettes and lots of leaves associated with them. Looking on the TAIR website (Arabidopsis.org) at these ecotypes’ pictures they appear that they don’t normally have aerial rosettes. I will have to do some further research into why my plants have them and if it is stress related.
I have also realized when I referenced other papers concerning Wa-1 and Landsberg ecotypes habitat range and apparent separation I found that they do indeed overlap. I will continue to do work with this tetraploid as well as the associated ecotypes.
I have had quite the challenge with stress experiments and my plants. I have been attempting many times this month to grow plants on various media with different concentrations of mannitol (osmotic stress), salt, and the combination of the two. The problem does not lie with the seeds germinating other than some bleaching or inviability of ecotypes (particularly CS22592) it is the bacterial and mold contamination. When this happens I generally throw out my plants. To combat this it was decided that plates will go outside of the growth chambers under the 24 hour light conditions. This will restrict the amount of close contact with soil, humid conditions, and constant air flow. This being said we have decided not to base stress experiments on plates alone but on seedlings grown on standard media then placed under stressful conditions.
I have attempted to do one such experiment with Wa-1 and its relatives and the Swedish ecotypes. The set up is as follows: grow seedlings on vertical plates until the appropriate size for planting, then remove them from their growing media and place them (roots primarily) on filter paper soaked with 250 mM NaCl or water (control) and continue to keep filter paper moist for 6 hours, finally plant the seedlings as normal in potting mix. I left these under the 24 hour light conditions. The salt treated plants all died and so did a large majority of the control plants. Obviously salt kills the plants but so does this process and I noticed that many of the seedlings leaves were dried out and mold was on a lot of the still living plants leaves (assumingly it grew on the sucrose on their roots). I will revise this experiment change the concentrations/time and continue to work on this during the month of August.
August
This month I have been continuing to observe the Swedish ecotypes that were placed under 24 hour light conditions. The ecotypes Stockholm and Henriksjfall (Hen) are starting to dry down and Skuleberget plants are starting to bolt. The tetraploids are still growing leaves and any inflorescences are not noticeable. Stockholm has more leaves, a larger diameter, and had more buds than Hen. They had similar bolting and flowering times though. The later flowering time of the tetraploids is evidence that they are similar to the classical definition of polyploids. Also I have been keeping track of the flower diameters of these ecotypes to see if the polyploids have larger flowers. In respect to seed size it does appear (via my observation and not by measurements) that the seeds of these tetraploids are bigger than the diploids. This is something I will measure if and when they produce seeds. These classic polyploid characteristics are seen in these Swedish ecotypes but are not seen in Warschau (wa-1) which is an interesting distinction.
I have also been collecting seeds from the ecotypes I have been comparing to wa-1. I have noticed that as wa-1 and Landsberg (lan) produced seeds and then started drying down they both produced more inflorescences. It appears that wa-1 produces more flowers than lan during this time. From this observation I plan on growing just lan and wa-1 and then comparing their life spans/reproductive success. I think that since wa- 1 produces fewer seeds per silique than lan then maybe it continues to produce flowers and seeds for a longer period to make up for this reduced fertility. Also the longer time period of seed production would reduce the possible hypothesized competition between these ecotypes because when Landsberg is finished with its life cycle wa-1 is still able to produce seeds. An ecotype with staggered seed production throughout the growing season could avoid some stressful conditions by producing seeds before or after the event. This could be a reasonable hypothesis because I have not been able to find a difference in fitness during salt and osmotic stress conditions for lan and wa-1.
As for stress experiments I transferred wa-1, cs994, cs22592, cs22593, and lan from either ms+sucrose or just ms to plates containing 170mM NaCl either containing or without sucrose in them. We wanted to see how these seedlings responded to the stress condition but also what effect sucrose had on plate experiments like this. I found that overall seedlings grew very poorly on ms plates alone and when transferred to the stress plates with or without sucrose they continued to do poorly and consequently died within 3 days. The seeds that were germinated on sucrose and then transferred to either sucrose or sucrose free 170mM NaCl plates lived longer than their ms counterparts. The roots were slightly longer and the leaves were not as bleached in the stress plates containing sucrose. Since they showed a small difference in fitness compared to the ms+salt plates I will continue to do stress experiments with the seeds germinating on ms+ sucrose and then transfer them to ms+salt stress conditions. This will remove any additional ‘help’ the plants are getting from the sucrose on the media. Since the plants became contaminated and also died quickly I am going to repeat this stress experiment with plates containing 130mM NaCl so I can take measurements on the plant’s response for a longer period time.
I am still looking into why the ecotype cs22592 has aerial rosettes which I expressed some curiosity about last month. I have read that later flowering plants generally have aerial rosettes controled by the ART 1 allele and that it often interacts with FLC and the FRI alleles (Poduska et al. 2003). Since cs22592 is late flowering I am assuming this is simply its normal phenotype but I am growing some under 24 hour conditions to verify this. I am curious about this because the ecotype cs22593 which is closely related to cs22592 does not have aerial rosettes. I am simply looking at these for curiosity’s sake and wondering if a stress (in my case fungus gnat infestation) could activate this allele.
Poduska, B., T. Humphrey, A. Redweik1 and V. Grbic, 2003 The Synergistic Activation of FLOWERING LOCUS C by FRIGIDA and a New Flowering Gene AERIAL ROSETTE 1 Underlies a Novel Morphology in Arabidopsis. Genetics 163: 1457–1465.
September
This month I have been continuing to monitor the growth and flowering times of the plants that are being compared to Wa-1. They are just starting to dry down and I have started to process and analyze the data from this experiment. Comparing the data from Landsberg, Landsberg erecta, and wa-1; wa-1 has the latest bolting and flowering of the three ecotypes, is intermediated in diameter, and had them highest amount of leaves. These differences are very subtle and the three probably (yet to be determined) are not significantly different. The other ecotypes in the experiment: CS 22592, CS 22593, CS 994, CS 970, CS 6044, and CS 998 all are quite different phenotypically from lan, ler, and wa-1 (bigger, later flowering, more leaves, aerial rosettes, etc). In conclusion as of today I still have not found a distinguishing phenotype (other than wa-1 producing fewer seeds) that differs between wa-1 and other ecotypes.
Last month I mentioned the hypothesis that wa-1 out lives Landsberg and by doing so it can withstand stressful conditions and produce an equal amount of seeds since it does produce few. I have been observing them and I basically found the same as the above experiment- that Landsberg bolts and flowers a few days before wa-1. Since they have just all flowered I will continue to monitor them and count seeds per silique, number of total siliques, as well as when they are done with their life cycles.
In August I had discussed some of the early results from the Swedish ecotypes that I was comparing under 24 hour light conditions. I had to throw them away because they were infested with thrips and they all began either dying or becoming infertile. I am going to try growing them again once we clean the grow chambers and the 24 light condition racks.
I have been observing wa-1 and the related ecotypes under stress conditions on plates that were transferred from MS+sucrose to the 130mM NaCl plates. Plates with this concentration of salt are wonderful for seeing differences in phenotype because the plants do not die quickly. So far I have noticed that wa-1 plants continue to grow roots for a longer amount of time than Landsberg and the others. I am removing the plants from the plates today and I will begin to quantitatively verify this observation. I am repeating this and I am also in the process of transplanting Swedish ecotypes to these conditions as well. Hopefully I will be able to find a trend in the results from this experiment to determine what makes the polyploids different from the diploids.
Earlier this month I realized that I had left some Swedish ecotypes in the refrigerator being vernalized since July 9 on plates containing 300mM mannitol and 250mM and 200mM NaCl. I placed them on the 24 hour light racks and all of the plants on the NaCl plates died right after they germinated but the plants on the mannitol plates survived longer. I found that diploid ecotype Stockholm had the greatest amount of leaves and that tetraploid CS 6183 had the same number of leaves as the control CS 6183 plate. Overall the ecotype’s roots on the stress plates were all similar and they were much shorter than their controls.
Also, I planted some of the control Swedish tetraploid plants that were very large on the plates. CS 6183 bolted and flowered after 12 days! This is very exciting considering that generally it takes over 100 days for them to bolt without long term vernalization. Whether it flowered early because they were so large on the plate, or whether it is because the seedlings were receiving continuous light, or the long vernalization , or some other condition is still yet to be determined. Perhaps going from continuous light when a seedling to long day conditions when planted with presumably less humidity in the chamber caused this ecotype to flower early.
My curiosity with the ecotypes with aerial rosettes has been increasing throughout this month and I am currently vernalizing CS 6044, CS 994, CS 970, and CS 22592 for 3 weeks to see if this will cause them to flower earlier and not grow any aerial rosettes. I also want to see if this phenotype is repeatable to determine if it’s the natural phenotype. There were many stressors in the growth chamber while these ecotypes were growing when I noticed this phenotype including high humidity, crowding, fungus gnats, powdery mildew, bacteria laden soil (from gnatrol), and over watering (application of gnatrol). I am going to grow these plants outside of the growth chambers, as well, to see if it does have something to do with those conditions.
October
October review
I finished a couple of experiments this month and I presented the results to the lab.
For the Wa-1 relative comparison experiment I found that Wa-1 is significantly different from the other ecotypes in the amount of seeds produced as well as in days to flowering. (I compared wa-1 to Landsberg, ler, CS 994, CS 22592, CS 22593, CS 6044, CS 3180, CS 998.) It seems that if Landsberg and Wa-1 do indeed live in the same locations and habitats then lan could out compete wa-1. Since we do not know conclusively if these two ecotypes overlap in their range it is hard for me to make any further conclusions on how they would interact.
As for the salt stress experiments performed on 130mM NaCl and MS plates: when these ecotypes were on ms + sucrose before being transplanted lan, CS 22592, and CS 22593 all were similar in their root growth patterns and CS 994 and wa-1 were also similar to each other. After being transplanted to the salt plates the ecotypes showed the same pattern as above. I had very large standard deviations from these experiments, so, I want to take my data and make box plots. I think this way I can find a more convincing patterns of phenotypes. It is apparent from the data that Wa-1’s roots did out grow landsberg’s by quite a lot. CS994, lan, and wa-1 all had individual plants that bolted and flowered as well. Considering the success in seeing phenotypes from the tested ecotypes I then decided to perform the experiment again and test the Swedish ecotypes as well.
For the second experiment the only thing that was different in my methods was that the plants were not on the ms + sucrose media for as long as the first experiment because of contamination. I found that on the ms + sucrose wa-1 and cs994 again were similar in their root growth and so were lan, CS 22592, and CS 22593. This is encouraging because I repeated that part of my results from the previous experiment. After I transplanted them to the same concentration of salt media I observed (I haven’t performed statistical tests yet) that the plants are not having the same phenotypic responses as the previous experiment. In fact this time many of the plants died within the first couple of days. This makes me think that perhaps I made the media wrong either in the first experiment or this time. I know that for the first experiment the amount of agar I put in the media was half of what it should have been, so I am thinking some of the salt could have leached out of the media. (A lot of liquid condenses in the bottom portion of the plates.)
The results for the Swedish ecotypes on ms + sucrose are that CS6183 and CS1535 are not significantly different from each other and CS3114, Hen, Skul, and CS1535 are also not significantly different from each other. It is interesting that the two tetraploid ecotypes (CS6183 & CS3114) are different in their growth patterns. I do not have any data yet from the transplant part of the experiment, because of the problems stated in the above paragraph. I am still going to try to work on the data I do have though.
I am in the process of repeating this experiment again. I have lowered the concentration of salt to 50mM, so I the plants live longer and I can get more data from them. I also fairly certain I made the media correctly this time with the correct concentrations of reagents.I also want to record plant survival and measure lateral roots and I have also increased the sample size significantly.
As for the aerial rosette issue I had with many of my ecotypes: I found out that the intensity of light within the growth chambers is very low and since these plants probably have functional FRI alleles I think the light issue could have been the cause of the aerial rosette ‘intensity’. Never the less I think these ecotypes are really neat so I still want to see if they will grow these rosettes under optimal conditions and under stressful ones too.
November
This month I have focused primarily on my experiments dealing with salt stress. I performed a lot of data analysis. As for my stress experiments, I transferred seedlings from MS + sucrose plates onto the plates containing salt media. I used Petri plates with a concentration of 130mM salt (NaCl) in MS media. I left sucrose out of the stress plates. The ecotypes used were CS1535, CS22593, CS22592, Lan, Wa-1, CS6183, CS3114, Hen, Skule, and WS. I placed these plates vertically in 24 hour light conditions in order to measure root lengths.
Three ecotypes, Lan, WS, and Wa-1, had one seedling each that bolted and flowered on the plates containing salt. These ecotypes are thought to be closely related so it is expected that they may have similar phenotypes. What is interesting is that Wa-1 is a tetraploid and it had the same phenotype as the diploids it is related to. I feel that the rest of the results I found from this experiment should be disregarded. I think this I had a small sample size after many of my plates were contaminated by bacteria/fungi. I am not confident in my results from my previous experiments either because there may have had contamination that I could not see with my naked eye that caused some of the odd phenotypes that I recorded. This holds especially true in the incredibly long roots wa-1 grew in the first experiment but I could not repeat in the experiments after.
In order to counter the problems I am having during these experiments I am going to start using square plates instead. This is in order to increase the area for root growth to occur. Also, this will allow me to take measurements for a longer period of time and I can increase my sample size as well. After allowing my seeds to break dormancy I plated the seeds on MS + sucrose and placed them vertically on the 24 hour light condition racks.
I placed the seeds from the Swedish ecotypes that I vernalized on plates for 8 weeks in a growth chamber. Ecotypes included were: CS6183, CS3114, Hen, Skule, CS1535, and two field collected populations from Huleplstjak and Al Stenar. I plan on planting these and comparing their bolt and flowering times, leaf number, plant diameter, and seeds per silique.
December
My progress on the salt stress experiments was slow this month because of the holidays. This being said though, I have found that growing the seedlings on square plates is better for performing these experiments. The seedlings grew more uniformly and contamination was not as widespread. I am repeating this experiment currently.
The Swedish ecotypes that I am growing in the growth chamber, presently, have all bolted and many so far have flowered. It is apparent from this that the tetraploids do require a long period of vernalization (8.5 weeks) in order to flower. This long vernalization did take a toll on the diploid ecotypes, and I had many plants that died after I planted them. I am also suspecting that thrips have made it back into the growth chamber. The infestation is minimal at this point and I am hoping my plants will not be too heavily affected.
