Plant Detectives Manual: a research-led approach for teaching plant science
Activity 10: Microscopy analyses
10.1) Introduction and objectives
Thus far you have phenotyped your plants by comparing external morphology, architecture and growth. By following the protocol below, you will use basic microscopic techniques to look at plant anatomy. The activity focuses on stem anatomy, but you may also choose to look at leaf and root anatomy. Allow your plants to be your guide in this exercise: if you can see a difference in a structure between your wild type and mutant plants, dissect that structure and endeavour to determine what has changed.
The objectives of Activity 10 are to:
- examine the epidermal morphology and vascular tissue
- compare any changes in wild type and mutant plant stomata number and aperture (i.e., how wide the stomata can open).
- microscope slides
- microscope (one)
- sticky tape (Sellotape)
- Petri dishes
- thin forceps (one–two)
- razor blades (one–two)
10.3.1) Examination of stem cross-sections
We will section stem pieces of Arabidopsis (of wild type and your mutant) using a vibratome. You will embed five-millimetre-long pieces of stems in 3% (w/v) agarose (provided) and make cross-sections of the stems. The use of the vibratome will be demonstrated in the class. The vibratome sections will be approximately 0.1 mm in thickness.
Transfer the sections to a glass slide and observe them under a light microscope. Compare the anatomy of the cross-sections of wild type and mutant; e.g., for total stem diameter, cell types and cell sizes, number of cell layers (Fig. 10).
10.3.2) Examination of root cross-sections (optional)
If your group has time and interest, you may also want to section root tissue. Ask your demonstrator for advice and then follow protocols described for stem tissue above. Note this may be tricky with thin Arabidopsis roots unless it has undergone secondary thickening and has a larger diameter than it did when it was a germinated seedling. Try this if you expect a root phenotype in your mutant.
10.4) Expected outcome
- Describe any differences of the stem cross-sections between phenotypes. Do stems of the wild type and mutant differ in size or structure? A drawing is a good way to illustrate the morphology of stem cross-sections. Label all structures that you can identify. Record the size of anatomical elements and describe other differences, if any (record the scale and show it on your drawing). Alternatively the stem cross sections can be photographed and put in your laboratory notebook.
- If it was observed, describe root cross-sectional anatomy and compare the two phenotypes. Do roots differ in thickness? Cell sizes? Arrangement of vasculature? Again, a drawing in your laboratory notebook is a good way to illustrate the morphology of cross-sections. Label all structures you can. Record size of anatomical elements and describe other differences, if any (record the scale and show on your drawing).
Figure 10: Cross-section of the stem from Arabidopsis
Micrograph showing a cross-section of Arabidopsis stem. vb: vascular bundles, xy: xylem, ph: phloem, co: cortex, ep: epidermis, if: intervascular region. Image from Turner and Somerville 1997.