Previous Next

Plant Detectives Manual: a research-led approach for teaching plant science

Activity 2: Seed germination and root growth

2.1) Introduction and objectives

Seed germination is controlled by genetic and environmental factors. Some, such as Arabidopsis seeds, require humid conditions and germinate only in the light. Other seeds will only germinate in the dark or following a series of moisture or temperature changes. Seed germination is regulated by hormones, specifically abscisic acid (ABA), which is an inhibitor of germination, and gibberellic acid (GA), an inducer. Mutations in genes that regulate germination could affect either its onset or timing. Seed germination and early development phenotypes are useful during mutant screening.

 

Arabidopsis seeds exhibit a two-step germination, in which the seed coat (testa) ruptures first, followed by the rupture of the endosperm by the radicle (Muller et al. 2006). The tip of the root consists of rapidly dividing cells; these divisions cause the initial (or primary) root to extend the longer axis of the root. After a few days, thinner, ‘lateral’ roots emerge radially from the primary root (Fig. 4B) and epidermal cells of the primary root will differentiate into root hairs, thereby increasing total absorption surface.

 

Image

Figure 4. Investigation of seed germination and root growth

A) Arabidopsis seeds were sterilised and plated in autoclaved agar medium supplemented with medium (MS salts). After germination under light, plates were placed vertically and root growth monitored. Photo of 12-day-old seedlings. B) Seedlings with primary root (pr) and lateral roots (arrows). C) Root tip and root hairs (rh). D) Gravitropic response of a root after being placed horizontally. Image A courtesy of Peter Crisp (The Australian National University); Image C courtesy of Humboldt University; Image D courtesy of Whitehead Institute (MIT).

 

Specific structures called statoliths, which are starch-accumulating amyloplasts, are present within specialised cells found in root tips (statocytes). Statoliths settle in the direction of gravity and are responsible for the ‘gravitropic response’ (Fig. 4D). This response senses the pull of gravity and directs root growth downward (Morita and Tasaka 2004).

 

You will monitor the germination, root growth and gravitropic response of seedlings grown on plates over the next two weeks. See more information about seed germination and root growth in the links provided in Appendix D: Databases and web resources.

 

The objectives of Activity 2 are to:

  1. determine the germination rates of wild type and mutant seeds as the percentage of germinating seeds relative to the total number seeds plated
  2. describe and quantify the gravitropic response of wild type and mutant seedlings
  3. contrast patterns of root growth and morphology between wild type and mutant seedlings.

2.2) Materials

Fig. 4A shows the experimental set up for Activity 2. Arabidopsis seeds were sterilised and plated in transparent Petri dishes containing autoclaved agar medium supplemented with Murashige-Skoog medium (MS salts). Seeds were stratified (incubated in the dark at 4 ºC) to coordinate germination to occur four to six days before being transferred to the light. After germination, plates were placed vertically and root growth monitored. A detailed protocol for the preparation of the plates is provided in Appendix F: Seed sterilisation and plating or click here.

 

The materials required for this activity are:

  1. growth chamber
  2. laminar flow cabinet, sterile seeds, filter paper, pipette, tips
  3. Micropore tape or parafilm
  4. wild type and mutant seeds (~ 50 of each)
  5. per group; four square Petri dishes with 20 millilitres of sterile 1.2% (w/v) agar and 0.5X Murashige-Skoog (MS) salts. Circular Petri dishes work, but can limit the number of seeds
  6. ruler
  7. sharpie marker
  8. scanner or camera to record plates (optional)
  9. styrofoam racks to hold plates vertically

Image

2.3) Procedure

Note that steps 1 to 5 will be performed for you in advance. You need to start from Step 6.

Materials

Method

MS agar plates; sharpie

1. Label four to six plates with ‘date, group name (A–E), plate (1–4).’

Ruler, sharpie

2. Trace one line with the ruler and sharpie at roughly 2/3 of the plate side length.

Laminar flow, sterile seeds, filter paper, pipette, tips

3. Using a long glass Pasteur pipette or regular-tip pipette, plate six sterile wild type seeds on one side and another six seeds of the mutant on the other side. If possible, randomly assign side of plate. Label with thin sharpie accordingly.

Micropore tape

4. Seal the plates with strips of Micropore tape or parafilm.

 

5. Stack the plates on top of each other, wrap with aluminium foil and incubate them in the dark at 4 ºC for five days to coordinate germination.

Styrofoam rack, growth chamber

6. Place the plates vertically on the rack provided and transfer them to the growth chamber with a light intensity of ~120–150 micromole (µmol) m–2 s–1. Plates can be incubated under continuous light or for a 16-hour photoperiod. TIP: make sure the light source is from the top and directly perpendicular to the plates during germination and early growth (before Step 10).

 

7. Record total number of seeds, and how many germinate after three days in the light.

 

8. Monitor root growth and development for two weeks.

Ruler

9. Follow root growth rate by marking the position of the tip (and only the tip) of the main root on the plate surface with a Sharpie at periodical intervals (two–four) days. Also note root direction (angle) and secondary/lateral root density.

 

10. Flip/rotate orientation of two plates 90 ºC after seven days, so that the growing seedling roots are now oriented horizontally. Over the next several days observe root orientation response. Measure root lengths before and after flipping (see Step 11).

 

11. Images of plates can be recorded with a scanner or camera. TIP: a scanner is usually more effective. Either way, place a black background behind the plate to increase the contrast of the white roots. Include a ruler as a scale. Note that the plastic lid will interfere with the camera flash or focus, to avoid this, plates may be imaged from the bottom face. Check image quality before proceeding with further recording.

2.4) Expected outcomes

  1. Record the number of germinated seeds per genotype to calculate the germination rate. We will score germination as the endosperm rupture evidenced by the emergence of the radicle through the testa after three, four and five days. If you record when each seed germinates you can also determine mean time to germination for each genotype in addition to total germination percentage relative to the initial number of seeds sown. You should count each plate separately and record data. You will then take the mean of the values across your plates so that you can do the appropriate statistics.
  2. Monitor the root length over time. This will allow calculating root growth rate as the change in length over time (millimetres/day). The easiest way is to measure the distance between the line on the plate and the tip of the primary root. More accurate and quantitative root analyses can be achieved by scanning or photographing the plates and using the free EZ-Rhizo software package (Armengaud et al. 2009) or ImageJ, as explained in Appendix E: Software tips. If you choose to do this, use your phone or a camera to take pictures of the plates — make sure to include a ruler on the side of each photo for scale.
  3. Describe and compare root morphology of the two genotypes (i.e., primary root, growth direction, and number of secondary (lateral) roots).
  4. Record any other observations that you consider relevant.
  5. Determine if there is a statistically significant difference in the above parameters between the wild type and the mutant (see data analysis Appendix B: What do I do with my data?).
  6. Remember to record your data in your spreadsheet and to record the plate number for each measurement you take.

 


Previous Next