By: Susan Siobhan Chen College Park Scholars-Life Sciences Program

1. My Internship Experience “Finding the Homozygous Double Mutant of the Duplicon Genes AtCel2 & AtCel4 in the flowering plant Arabidopsis thaliana” By: Susan Siobhan Chen College Park Scholars-Life Sciences Program University of Maryland, College Park schen@umd.edu

2. Introduction I worked in Dr. Elena del Campillo’s lab at the University of Maryland, College Park. Her lab is part of the Department of Cell Biology & Molecular Genetics located in H.J. Patterson Hall (pictured above). This lab is part of the Arabidopsis thaliana Research Initiative at the University of Maryland (ATRIUM). Arabidopsis thaliana is a very good eukaryotic model system for genetics research and is studied all over the world.

3. Work and Accomplishments Overview: • Abstract/Introduction • Materials • Methods • Results • Discussion • Unexpected Events and Situations • Observations About My Experience • Acknowledgments

4. Abstract/Introduction: • Cellulose and cellulase both play important roles in plant growth and especially plant cell walls. We investigated the cellulase genes AtCel2 and AtCel4 in the flowering plant Arabidopsis thaliana. These genes are duplicon genes and may have similar roles and may have evolved similarly. We are searching for a homozygous double mutant plant which does not have both the Atcel2 and Atcel4 mutant genes present. We determined if the plants had the Atcel2 and Atcel4 genes by doing PCR and DNA analysis. So far, we have found that we have plants that are heterozygous for the double mutant genes. We will then self-pollinate these plants and hopefully find the homozygous double mutant. We also analyzed how the mutant Atcel2 and Atcel4 plants grew differently compared to the wild type plant. We observed the kinetics of the growth of the different plants by measuring the root length of the plants in different medias-(control and sucrose medias).

5. Arabidopsis thaliana plants- we planted these in soil or placed seeds on media plates and observed growth. Media plates- to grow plants Microscope- to examine plants Grinder- used to grind plant leaves to extract DNA Techne Genius machine-used to run PCR reactions 10X PCR Buffer Thermo Pol, 25 mM MgS04, dNTPS 2.5 mM each, Primer FW728 (12uM), Primer LBb (12 uM), Water, new Taq NEB, gDNA- all used in preparation for PCR reactions Gel electrophoresis machine-used to run gel for DNA analysis Nikon Coolpix camera- used to take pictures of plants for analysis of root length and other growth observations Image J program from NIH- used to measure plant growth Materials

6. The following are steps were used in a protocol to determine if plants had the Atcel2or Atcel4 gene: Isolated DNA from 13 individual double cross plants generated by crossing mutant cel4 female with mutant cel2 male. Also isolated DNA from one wild type plant. DNA isolation according to Edwards protocol Resuspend DNA 100 ul of water So tested 13 double cross plants, one wild type for negative control and the corresponding homozygote for positive control Polymerase Chain Reaction (PCR) for DNA from all plants Run gel electrophoresis to analyze DNA of plants Take picture of gel and analyze Other tasks I performed included: Planting Arabidopsis thaliana plants Taking pictures of Arabidopsis thaliana plants and observing root length growth Measuring root length growth using the Image J program Methods

7. Results • So far, we have confirmed that we have the heterozygous double mutant plants which have the Atcel2 or Atcel4 genes. Through our analysis of root length and growth, we observed that the Atcel2 plants grow the fastest in sucrose and the Atcel4 plants grow the slowest in sucrose. Control Plate Sucrose Plate

8. Results (Continued) Note: The average of the length of 10 plants was measured. The program Image J was used to measure the root lengths.

9. Results (Continued) Note: The average of the length of 10 plants was measured. The program Image J was used to measure the root lengths.

10. Discussion We believe that the mutant Atcel2 plants grew faster in sucrose because the mutant Atcel2 plants have a defective Atcel2 part. However, the Atcel4 part of this plant is normal and will try to make up for this and therefore the roots grow faster. The Atcel4 plants have a defective Atcel4 part, but the normal Atcel2 part is not as expressed in the roots so the Atcel4 mutant plants grow the slowest. We are still looking for the homozygous double mutant. We do not know the phenotype of this plant, but we hypothesize that it will be different than the wild type plants since cellulases play an important role in plant growth and the homozygous double mutant plant would have defects in 2 of its cellulase genes.

11. Unexpected Events and Situations • When we were observing the phenotypes of the plants, we noticed that some plants had wrinkled leaves. We then discovered that there were transparent bugs living in plant, which may have made the leaves wrinkly. • Sometimes the DNA bands did not show up when we ran the gels.

12. Observations About My Experience Through this internship experience I gained lots of knowledge of how research in a lab works. Some of the techniques I learned were basic and important for work in any lab. This was what I expected. But I also learned so many new techniques and so much information about Arabidopsis thaliana plants that I had never known about before. Being a pre-medical student, I know that I will be doing research in the future and this lab has given me a strong background and foundation for my future endeavors. I would do this experience again since I learned so much about research and life in such a short time.

13. Acknowledgments I would like to thank my mentor, Dr. Elena del Campillo for letting me work in her lab and teaching me so many research techniques and how to be a real scientist! It was truly a wonderful, enlightening experience. I would also like to thank my lab partner, Ana for teaching me and helping me in the lab. I would like to thank the College Park Scholars Life Science program, and especially Dr. Lee Hellman and Stacy Richardson for giving me this amazing opportunity to learn so much and explore the field of research. Last, but certainly not least, I would like to thank my family for their never ending love and support.