Repotting, Re-PCRing, and the War for the Greenhouse

Life has been hectic since returning to campus for summer research. I dove back into work with a massive repotting of my milkweed. My project requires two stems of milkweed in every pot—on one stem I will simulate insect herbivory, and I will measure cardenolide levels in both to determine whether chemical defense signals are shared through root systems. Splitting and repotting milkweed is an all-day event, mostly because by the end of it you’ll be too dirty and exhausted to want to do anything else. Milkweed naturally occurs in fields and prairies, where plants send out sprawling root networks that take up water and nutrients and anchor the plants so they can grow as tall as over two meters. Though our plants in the greenhouse never grow that tall, their roots still try, and milkweed pots are crammed with tangled and gnarled knots of them. My labmates pitched in to help me out on repotting day, which I will always be thankful for, considering what a huge and messy task it was! We shook out gallons of loose dirt and chisel away more from the roots, so we could trace roots to see which stems are closely collected and can become a pair in its own pot. We do all this while trying not to snap the delicate stems, which grow to about two to three feet in the ISC. By the end of the day, we were all covered head to toe in potting soil, as were the floors of the greenhouse, which we bleached and scrubbed.

My plants have to “rest” before I stress them out any more, so for the past few weeks I’ve been working on the milkweed microsatellite project that Hannah Call, Katie Stahl and I have been collaborating on since Fall 2016. We’re extracting and amplifying DNA from wild populations of milkweed that we have trait data for (such as height and number of leaves) as well as spatial data (where the stems are in relation to one another in space.) We want to examine genes called microsatellites in the plants to determine which ones are clones and which are genetically distinct. This will let us relate genetic distance to the trait and spatial data to determine whether relatedness can predict traits, and if distance can predict relatedness. Katie collected the tissue samples last summer, and since then we’ve been working on amplifying the microsatellite stretches of DNA, but we got some bad news at the end of last semester. Our DNA extractions, which we use to perform the amplifications, have degraded beyond the point of usability. We’re hoping that the genotyping we’ve already done is still accurate, but in order to check, we have to re-extract the DNA from the original tissue and perform polymerase chain reactions (PCR) on the extractions again. It’s frustrating, because Hannah and I spent the entire 2017-2018 school year doing the original PCR that we may have to scrap, but we’re really hoping the duplicates come back clean and we can keep moving forward with the project!

In general plant lab news, the greenhouse has been overrun with several pests! Everyone in Puzey Lab marched off to do war with the thrips and spider mites last week, armed with sachets of predatory mites to eat the thrips and mysterious vials of wood chips that will, somehow, deter the spider mites. We hung the mite packets on the milkweed like Christmas ornaments, and scattered the wood chips on the leaves like glitter. All in all, a very festive experience! Since then we’ve still had to use manual methods of pest management, such as wiping down the leaves, spraying bleach and hand-washing the plants with insecticidal soap. This last method has left me sopping wet as I write this post—hopefully we will triumph soon, and face drier days ahead.

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