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Brad, shown above freezing leaves with liquid nitrogen, worked on getting RNA out of idioblasts. By the end of the summer we were increasingly frustrated. We decided to take a step back and look for the best method for getting RNA out of Dieffenbachia leaves. Once we had that, we planned to test it on isolated idioblasts. Off to the library went Brad to track down as many possible methods recommended for recalcitrant plant tissues. Brad returned to the lab in the spring semester to try the methods he'd found. By the end of spring we had excellent recovery of pure RNA from leaves. Now, if we can only get this to work for isolated cells.
Meanwhile, Bob continued to try for proteins from idioblasts. His purple tubes from protein assays proved he had proteins, but electrophoresis gels showed only some faint bands. He continued doggedly through the summer and fall semester. He learned a lot about the problems and obtained results that were suggestive, but we weren't convinced they were plant proteins and not Bob's.
In the summer Richie volunteered for a few hours a week and tried to isolate the crystals for chemical study, but that proved a tough project. Getting idioblast cells away from other cells is easy precisely because they are tough cells. Smash everything else and they survive! Once you have them, though, it is hard to get them open to get the crystals out, and Richie never did get enough to study before the summer ended. Richie graduated in December 2005 and went off to New York.
Randy (shown clowning around with Dieffenbachia plant above) joined the lab in the Fall semester 2005. He decided to look at the development of idioblasts as the leaves develop. During the summer Brad did some work on this, embedding developing leaves in wax and making beautiful thin sections. Randy tried a different approach, doing mini-isolations of tiny developing leaves and cataloging the crystals that formed. The results show that crystals are forming from the earliest stages.
In the Spring semester 2006, Mary joined us. She took the procedure we had worked out for quick isolation of idioblasts and modified it to work with sterile technique. She hoped to get cell cultures for further study. Unfortunately she cultured more fungi than cells. To our surprise, she obtained cleaner cultures on the open lab bench than she did working in the laminar flow sterile hood. This project was put on hold until the department can obtain a dedicated tissue culture hood, but, thanks to Mary we now have a procedure ready to use when we got such a hood.
Bob, Brad and Mary all graduated in the spring of 2006.