Gravitropism: the Role of Roots
Technical & Teaching Notes
Introduction and context
The majority of plant tropism experiments examine the shoots, yet over 50% of the plant is below the soil.
This experiment using agar plates and white mustard seeds allows students to visualise the development of roots, their root hair cells, observe the zones of elongation and differentiation and illustrates gravitropism very effectively in roots.
This resource includes three extension activities, including visualisation of xylem in the roots, the effect of an obstruction in the ‘soil’ in the path of the root, and utilising graticules, a key skill in A Level Biology. There is also the option for students to watch video extracts from a lecture by scientist Prof Malcolm Bennett.
Safety Notes
Preparation of agar plates requires an autoclave using high temperature and pressure.
Used agar plates should be autoclaved before disposal.
6% bleach is used to sterilise the mustard seeds; safety glasses should be worn.
Apparatus
For each student/student pair:
· 1 pre-prepared agar plate (1% agar)
· White mustard seeds (3-4 per plate, allow a few extra in case they are lost during sterilising)
· 6% bleach, 50cm3 in a 100cm3 beaker
· 3 autoclaved distilled water aliquots in small glass containers (to rinse the sterilised seeds free from bleach)
· Forceps
· A square drinks bottle (such as an old squash bottle with square sides. Round ones such as a used bottle of fizzy drink is OK but needs propping up to stop it moving so is less preferable), with holes cut out the width of petri dishes to keep the agar plates upright
· Clingfilm (to wrap the agar plates preventing desiccation)
For activity task 1:
· Graticule
· Microscope
For activity task 2:
· A sterile obstruction eg lego block, glass beads, ball bearings
For activity task 3:
· Food colouring (blue worked better than red, green or yellow)
· 2 Cocktail sticks
· 250cm3 beaker
· Microscope slide
For activity task 4:
· No extra equipment
Technical Notes
1. Agar plates need to be prepared in advance of the practical; ideally within the same week, to decrease the chance of contamination of the agar.
1. Prepare 1% agar plates by mixing 10g/L agar (or BactoAgar) with distilled water in a bottle/flask.
2. Autoclave for 15 minutes at 15psi, 121oC.
3. Allow agar to cool to 40oC before pouring agar into sterile petri dishes to a thick depth.
4. Ideally, pour the agar in a hood to reduce contamination.
5. Allow agar plates to set, and store in a fridge until required.
(I did not observe any difference in germination or response of seedlings when using 1% agar with Murashige and Skoog (MS) mineral salts so have excluded these from the method.)
2. Sterilisation of white mustard seeds. You could use other seeds, however I found white mustard seeds better to work with than cress seeds. You could also use the model plant, Arabidopsis (see notes at end under ‘further investigations’).
1. Place the mustard seeds in a beaker with 6% bleach (6 cm3 in 94 cm3 of distilled water) for 8 minutes.
2. Remove the mustard seeds using sterile forceps and transfer to distilled sterile water.
3. Transfer the seeds after 20 seconds to fresh distilled sterile water, and then give one more rinse following this. The 3 rinses in water will ensure that all bleach residue is removed.
3. Planting of sterile mustard seeds onto agar plate
1. Using sterile forceps, transfer one mustard seed at a time to the top third of the agar plate.
2. Gently press the seed slightly into the agar. It should not be buried, but you don’t want the seed to slide down the agar when the agar plate is held vertically. 3 or 4 seeds can be placed on each agar plate (figure 1).
3. Wrap each plate in clingfilm, to prevent desiccation and then place the agar plate in the vertical position in the cut plastic bottle (figure 2).
4. Leave seeds to germinate: a window sill or shelf is ideal.
5. Agar plates should be observed daily to monitor growth. Growth of roots typically occurred within 24-48 hours and at 48 hours was long enough to proceed to the next step.
Figure 1: example of planted Figure 2: a cut drinks bottle to hold agar plates
seeds on agar
Activity 1 – Observation of root hair cells, zones of elongation and differentiation and measurement of root hair cells with graticules
At 48-72 hours there should be visible root growth from the mustard seedling to enable root hair cells to be observed under the microscope (figure 3).
The clingfilm should be removed and the agar plate without the lid can be placed on the stage of a microscope to observe the root hair cells.
Students were amazed at the number of the cells and viewing them in 3D rather than a diagram (figure 4). Students can easily take pictures using a camera or mobile phone held up to the eyepiece of the microscope.
Figure 3: Roots on mustard seedlings Figure 4: root hair cells
at 48 hours
Using the microscope, the students should easily be able to identify the zone of elongation where mitosis occurs in the root tip and then the zone of differentiation where the root hair cells begin (figure 5)
.
Using a graticule placed on top of the root, students are able to measure the length of the root hair cells. It is better to place the graticule onto the agar than move the roots onto a microscope slide, as the root hair cells become distorted and bent.
Questions such as ‘the further the distance from the zone of elongation the longer the root hair cells’ or ‘Is there a ratio between width of root and length of root hair cells?’ could be asked.
Activity 2: Extension task – to observe the response of roots to an obstruction in their path
This models the effect of an obstruction, such as a stone, in the soil that roots might encounter, by placing an obstruction ahead of the root in the agar. I used sterilised glass beads, but a sterile lego block, ball bearing or other such object could equally be used. This allows students to observe what happens when the root senses an obstruction and changes its growth which would not normally be visible below the soil.
Activity 3: Extension task – to observe the xylem in the roots using food dye
This is a good extension task that students can undertake after observing the root hair cells and the zone of differentiation to visualise the xylem by placing the seedlings into water coloured with food dye. Blue food dye worked the best.
Students need to remove the seedling from the agar using forceps and suspend the seedling on 2 cocktail sticks over a beaker of water containing a couple of drops of food dye. The root hair cells need to be below the level of the water. Leave the seedling for 24 hours. Remove the seedling and place onto a microscope slide and focus on low or medium power to reveal the xylem which will be coloured with the food dye. The water is taken up by osmosis by the root hair cells to enter the xylem.
Figure 7: suspended seedlings in coloured water Figure 8: root and root hairs with xylem dyed blue after water uptake by osmosis
Activity 4: Extension task – to observe gravitropism in mustard seedling roots
Most tropism experiments are on shoots but with the seedlings grown on agar it is easy to observe gravitropism in the roots simply by rotating the agar plates. This is a simple extension task that could be done on remaining seedlings on the agar plate after observing root hairs, the xylem, or the effect of an obstruction.
Background information
Plants respond to gravity - gravitropism. In roots, this is positive gravitropism and they grow towards gravity and deeper into the soil. This helps anchor the plant, as well as allow roots to obtain water and mineral ions. Water is absorbed by osmosis through the root hair cells of roots to then enter the xylem to be transported throughout the plant. (See our animation on plant transport: http://www.saps.org.uk/secondary/teaching-resources/1274)
In the soil, plant roots will naturally meet obstructions such as stones, hard lumps of mud which the roots can sense and change the course of their growth.
This practical was devised after attending a lecture by Professor Malcolm Bennett of the University of Nottingham on Root Biology, “What happens below ground? A multidisciplinary approach to Root Biology”. His lecture is available to view freely at: http://www.gatsbyplants.leeds.ac.uk/tree.2.0/gatsby_tree.php?theme=null
It has some great video footage showing gravitropism in roots and links to the statoliths that sense the change in gravity; this would be an excellent extension activity for students following the practical.
Further Investigations
Arabidopsis (the model plant that has had its genome sequenced) is an alternative seed that could be grown for these experiments.
Arabidopsis seeds are available free for schools through the European Arabidopsis Stock Centre at the University of Nottingham. (http://arabidopsis.info/CollectionInfo?id=49).The ‘Plant Curiosity Kit’ contains natural mutants (not genetically engineered) suitable for use in schools. This activity could be linked to examining the mutations in the DNA.
Acknowledgements
This practical was devised by Dr Iona Martin of Colchester Country High School for Girls after attending the SAPS Gatsby Plant Summer School in July 2013, funded by an ENTHUSE Award.
The resource was inspired by Prof Malcolm Bennett of the University of Nottingham.
Science & Plants for Schools: www.saps.org.uk
Gravitropism: the Role of Roots: p. 1
Revised 2013