1. Comparison of trichome spacing between Columbia wild type and P80 mutant Arabidopsis thaliana using scanning electron microscopy
Image taken by researcher
Jacob Jaminet
Central Virginia Governor’s School for Science and Technology
Class of 2013

2. Background: Arabidopsis thaliana
Arabiposis is a plant
Its entire genome has been mapped
Able to turn genes on/off
Able to study differences between mutants with the Scanning Electron Microscope (SEM)
millar.bio.ed.ac.uk
wistep.wisc.edu

3. Background: Trichomes
Single-celled epidermal cells
Protect plants from insects and water loss
This project studied the spacing, distribution, and formation of trichomes on leaves of Arabidopsis
bms.brookes.ac.uk
Image taken by Researcher

4. Trichome Development (Hülskamp, 2000)
Schwab, Folkers, Ilgenfritz, Hülskamp, 2000
Microtubule skeleton
Spatial organization
Role of actin
Cell elongation

5. Previous Projects Primary vs Secondary leaves
Trichomes are hard to see because the leaves are curled around.
BSE
40Pa
Hill & Jannah, 2009

6. Introduction
Rationale: To study cell differentiation to explore the best ways to identify trichome development
Research Question:
What are the best conditions to image trichomes on Arabidopsis thaliana?
What are the differences between wildtype and P80 mutant Arabidopsis?

7. How does the SEM work?
Hitachi ppt, 2006

8. Backscatter Electron Detector
Objective Lens
SE Detector
High Energy BSE (>50eV) travel in a straight line towards detector
BSE Detector
Specimen
Hitachi PPT, 2006

9. Introduction (continued)
SEM factors
Accelerating voltage (kV)
Electron beam penetration
Probe current
Spot size of the viewing area
Object aperture size
Depth of field, current, and resolution
Working distance
Distance from objective lens to specimen
Variable Pressure
Controls the pressure inside the chamber from Pa – 270Pa

10. Materials and Methods
Seeds retrieved from Virginia Tech Fralin Life Science Institute
Seeds were surface sterilized with a 30% hypochlorate solution
Plants grown in Murashige-Skoog agar under sterile conditions using Labconco flowhood
Image taken by Researcher
Image taken by Researcher
Image taken by Researcher

11. Materials and Methods (Continued)
Stub prepared
Imaged with Nikon COOLPIX 950
Image taken by Researcher
Image taken by Researcher
Image taken by Researcher

12. Materials and Methods (continued)
Imaged with Nikon COOLPIX 950 attached to stereo microscope
Image taken by Researcher
Image taken by Researcher

13. Materials and Methods (continued)
Placed in Hitachi N3400 variable pressure scanning electron microscope
Image taken by Researcher
Image taken by Researcher
Image taken by Researcher

14. Results
See trichomes

15. Results (continued) Image Session Voltage Vacuum SE or BSE Result WT
10kV
40Pa SE
Hard to distinguish trichomes from leaves
P80 (1)
10kv
Same as above
P80 (2)
BSE
Faded images
Very hard to distinguish
P80 (3)
0Pa Se
Clear images
All the data from the photos appeared to be very much the same. There might be some data missing from the earlier photos we took during the 2nd six weeks SEM Senior Sem Friday.

16. Results (continued) Differences between P80 and WT P80 branches
Image taken by Researcher
Image taken by Researcher
Image taken by Researcher

17. Discussions and Conclusions (continued)
Gene Differences Groups (Schwab, Ulrike, Ilgengritz, & Hülskamp, 2000)
Trichome differentiation mutants
Endoreplication mutants
Branching mutants
Phase growth
Cytoskeleton

18. Results (continued)
Base

19. Discussions and Conclusions
Technique for imaging
Use petiole to arrange leaf
Place leaf so vacuum keeps trichomes upright
Best trial was after plants were studded then placed in the refrigerator for 10 hours
Image taken by Researcher
Image taken by Researcher

20. Refrigerated v. Not Image taken by Researcher

21. Backscatter Electron Detector
Do NOT use it
Makes pictures appear faded
Hard to determine trichomes

22. Backscatter v. SE BSE SE Image taken by Researcher

23. Recommendations Preparation Conditions Place leaves on stub
Put in a petri dish with cover
Refrigerate for ~8-10 hours
Then image with SEM
Conditions
10.0kV SE
40 Pa
Probe Current of 10.5mamp

24. Future Studies
Identify the carbohydrate whether it is cellulose or lignin for possible product development.

25. Acknowledgements
I want to thank Dr. Lindeman for her continued support during this project as well as Dr. Glenda Gillapsy from Virginia Tech for her help in supplying Arabidopsis and her help with background information of the plants.

26. References
Schwab, B., Ulrike, F., Ilgengritz, H. & Hülskamp. M., (2000). Trichome morphogenisis in Arabidopsis. The Royal Society, 355, doi: /rstb
Hill, J. and Jannah, N. (2000). Images of trichomes.
Hülskamp, M. (2000). Cell morphogenesis: How plants split hairs. Current Biology, 10(8),