WEEK 2: Ready… Set… Grow!

By Taylor Lovelace, Elizabeth Nightingale, Clair Stump

First just a couple definitions… these may be important in understanding what we’re talking about later on!

Arabidopsis thaliana: a flowering member of the mustard family, and the star of this show.

Genotype: The genetic make-up of an organism.  (In this experiment, 11 different genotypes of the plant Arabidopsis are used.)

Randomization:  A way to account for potential bias within an experiment.

Replication:  More is more!  The more specimens within an experiment, the better the results because we can account for any potential variation.

Planting our seeds!

Planting our seeds!

By week 2, thousands of Arabidopsis seeds (yes, we did say thousands!) had been sowed, refrigerated for a week, and were ready to be planted in the greenhouse. Earlier in the day, pots had been filled to the top with soil and were first watered to eliminate the risk of washing away the very tiny seeds.

Before we tell you what and how we did it all, first we want to tell you a little bit about why we did the planting the way we did… Each student was given a very specific schematic of where to plant each individual plant within the pot. Why you ask? Well it all has to do with randomization. Any good experiment needs to consider not only randomization, but also replication. These two aspects of an experiment are very important things to take into consideration.  Within any experiment, there will be variables that are beyond the control of the researchers that may influence the results.  In order to accommodate for these variations, it is important to randomize and repeat the treatments of all specimens that are a part of the study.  Here is a link to a video that explains the reasons for randomizing very well:  https://www.youtube.com/watch?v=Mufk0ZJVqQY

Let’s talk randomization for a moment…. Our goal in this experiment is to see how a change in a single factor (water) affects the growth of Arabidopsis plants. However, there are subtle, but important differences within greenhouse that can influence the growth of the plants. And, the plants can influence the growth of the others plants in the same pot. Crazy, I know! This is why we randomized where in the greenhouse the pots go and where we plant the seeds in each pot. So every genotype gets to try out every area in the greenhouse and also gets a chance to be a neighbor to every other type of genotype. Now to try all these different combinations we need a lot of seeds and pots. This is where replication comes into the story. We need multiples of each pot with all the different planting orders to put in the different zones of the greenhouse. This also helps account for inevitable die off. By planting a lot and I mean A LOT of seeds and by randomizing the plants, we help to ensure that our treatment variables are not confounded, it helps us be confident in the data we spend so much time and effort to collect.

Filling our pots with soil

Filling our pots with soil

Seeds from all 11 genotypes sown onto filter paper ready to be planted.

Seeds from all 11 genotypes sown onto filter paper ready to be planted.

And heres how we did it

Labeling all our randomized pots!

Labeling all our randomized pots!

Step 1: Each of us, or a pair of us, took a block (group of pots) that we were specifically responsible for… .no pressure! Each numbered block was made up of multiple trays and each tray contained 15 pots.  We created individual pot ID tags for each pot, and placed them according to its randomly assigned location.  On each tag we wrote the genotype, treatment type (high water or low water), the replication number, and the block number.  It was also important to add an extra identifying tag and different colored toothpicks for pots that contained 6 plants, other pots only had one plant and did not require these tags. By adding these tags it will make it easier to identify the plant’s genotype in the future since a plant’s location was also randomized within each pot.

Step 2:  Next, small indentations were made in the soil to indicate where seeds would be placed.  Using the germinated seeds prepared in Week 1, each wedge of filter paper with a single seed was placed on top of each indentation according to the randomized location.  It was very important to make sure that each ID tag corresponded with the correct and seed genotype was correct and placed in the right spot. This step was critical and required each student to be very meticulous in checking each other’s work!

Double checking each other!

Double checking each other!

Filter paper wedges each with one tiny seed, placed in the correct location in pot. Colored toothpicks remind us where we randomly placed each genotype in the pot.

Filter paper wedges each with one tiny seed, placed in the correct location in pot. Colored toothpicks remind us where we randomly placed each genotype in the pot.

Step 3:  Finally, after double checking to make sure each seed’s location and ID was correct, it was finally time to plant the seeds! Here, each student took a wash bottle and rinsed every seed off its filter paper and into an indentation. This process turned out to be quite hard with the seeds being so tiny but hopefully every seed made it into the right spot!……..  And after a few hours, planting was actually complete!  Whew! Now we get to sit back and watch them grow (hopefully in the right spot)!

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Washing seeds off the filter paper into the pots.

Washing seeds off the filter paper into the pots.

Everything is planted!

Everything is planted!

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Week 1: Seed sowing for cold treatment

By Rebecca Kim and Jeff Kimmel

This first week was all about careful preparation. In order to prepare for successful planting the following week, the first step of our experiment was to sow the Arabidopsis seeds to wet filter paper and have it undergo cold treatment for 1 week. Cold treatment is an important step of our experiment because it helps to improve the rate and synchrony of germination. Cold treatment ensures that all of our seeds will germinate at the same time, which is vital to our experiment. In addition to undergoing cold treatment, this first week of sowing seeds onto filter paper helped to alleviate the daunting task of planting almost 4000 seeds the following week. This task required keen eyesight, steady hands, and a lot of patience.

To begin, we divided our class into groups. Each group was in charge of a different genotype. Each group was given the same set of tools necessary for success: filter paper, paintbrushes, petri dishes, wash bottles, water, ethanol, markers, and of course lots of tiny Arabidopsis seeds! After acquiring the necessary materials, the next step was to label all of the petri dishes we needed with our assigned genotype. Then we cut each filter paper into 6 equal wedges, being careful not to cut all the way to the center (this way the filter papers could remain intact during cold treatment. Then when it comes time to planting, each wedge containing a seed can easily be separated when wet, and placed in the correct pot). After the filter paper was cut, each wedge was labeled with our assigned genotype (A-F, P1-P5). Then, we lay the filter paper in a petri dish and wet the paper with just enough water so that the paper would stick to the dish without an excess amount of water in the dish. This process also helped with keeping the Arabidopsis seeds in place.

Now we were ready to start placing the seeds on the filter paper! Each group had 2 beakers, 1 filled with water and 1 filled with ethanol. First we dipped our paintbrushes in ethanol to sterilize the brush, then in water. Using our wet paintbrushes we carefully brushed one seed onto each filter paper wedge. This was where the keen eyesight and lots of patience came into play… The seeds were VERY small! In between every few seeds, we dipped our paintbrushes in ethanol and then again in water, just to be safe. Finally, we covered the petri dishes containing 6 seeds each in saran wrap. This helped to prevent the filter paper from drying out during cold treatment. Finally, the petri dishes were placed in a cold incubator between 3-5°C for 1 week. After 1 week in the incubator, the seeds will be ready for planting!

Here our class is divided into groups with all of our supplies laid out. We are ready to tackle these seeds!

Here our class is divided into groups with all of our supplies laid out. We are ready to tackle these seeds!

Lizzie is demonstrating how we cut the filter paper into wedges and labeled each wedge with our assigned genotypes.

Lizzie is demonstrating how we cut the filter paper into wedges and labeled each wedge with our assigned genotypes.

We are bent over in concentration trying to maneuver these tiny seeds to get in their right place!

We are bent over in concentration trying to maneuver these tiny seeds to get in their right place!

When does plant diversity matter?

By Cynthia Chang

Plant communities provide important ecosystem services to us, such as food and natural resources. The more productive an ecosystem is, the more food or resources are available for us to use.  Having more plant diversity in a community is thought to increase overall productivity. The idea behind this is that different types of plants occupy different niches– or resource requirements. For example, some types of plants may have shallow roots while other types may have deeper roots. If you have a diverse plant community with many different depths of roots, the community as a whole is able to access more water along the entire depth of the water column. People have been applying this theory for a long time in agricultural practices, growing different types of crops together in hopes of maximizing productivity.

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Ecologists have been testing whether having more plant diversity increases ecosystem productivity in a variety of experiments with varying results. Recently, there has been a growing interest in understanding whether or not increasing genetic diversity within a single plant species increases productivity. The idea is that a genetically diverse group of plants will occupy different niches whereas a group of clones, genetically identical individuals, will occupy the same niche and thus, be less productive overall.

Our UW-Bothell Plant Ecology class is conducting a greenhouse experiment growing 3500 (!!) Arabidopsis thaliana plants to determine whether plant diversity impacts community productivity. We will grow 6 plants together in a single pot to represent a small plant community. Some pots will have high genetic diversity, while other pots will have lower genetic diversity. In addition, certain plant individuals will also be more genetically predisposed to exhibiting different traits under different growing conditions (also known as phenotypic plasticity). Some pots will have individuals that exhibit very different traits under different growing conditions (high phenotypic plasticity), while other pots will have individuals that always exhibit the same trait (low phenotypic plasticity). We will grow these plant communities under two different water conditions, high and low water, to see how different plants respond. At the end of the experiment, we will compare total biomass of each pot between all the different communities to determine whether more diversity increases productivity.

This project is part of the unPAK (undergraduates phenotyping Arabidopsis knockouts) and the data we collect will contribute to a nationwide, collaborative network. The goal of this network is to get undergraduate students involved in developing a database to help understand the genotype-phenotype relationship of different Arabidopsis mutants.

Check back in next week to hear more about our seed sowing adventures!