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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