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

Activity 1: Observing the plant phenotype

1.1) Introduction and objectives

A common and simple way to assess the effect of gene mutations is to observe the resulting phenotype. The phenotype is the expression of the underlying genetic information and reflects the interaction between genes and the environment. Describing the phenotype of your wild type and mutant plants over the course of their development improves your ability to infer the biochemical pathways or physiological processes affected by the mutated gene.

 

Plant growth analysis has been defined as an explanatory, holistic and integrative approach to interpreting plant form and function (Hunt et al. 2002). The classical, destructive method involves tissue harvesting at regular intervals and estimation of different physical parameters, such as dry weight of plant parts (leaves, stems, roots and reproductive structures), leaf areas and volumes. These primary data are then used to calculate other parameters, such leaf area index, root/shoot ratio, and relative growth rates, in order to study plant morphology and function. ‘Destructive methods’ are widely used and are effective. They are resource (i.e., many plants are required) and time intensive, however, and will only be used towards the end of the Plant Detectives Project (in Activity 9, when relative water content is measured).

 

An alternative method to study plant growth and development is the use of non-invasive techniques. These are based on the direct measurement of leaf number, size and height, or on periodical image recording. Measurements are performed under controlled conditions and often involve subsequent software analyses. Image-based growth analysis is becoming a common approach as advances in robotic technology become broadly accessible. Image analysis has the advantages of requiring less starting material than the classic destructive method and also enabling a given individual to be monitored throughout its life cycle. Moreover, the photographic record can be analysed at any time in different ways so as to look for other morphological traits.

 

The main goals of Activity 1 are to:

  1. follow the growth and development of wild type and mutant plants for several weeks. You need to familiarise yourself with the general morphology and organs of Arabidopsis (Fig. 1C) and the developmental growth stages described in Appendix C: Arabidopsis growth stages
  2. use the LemnaTec Scanalyzer 3D (LemnaTec, www.lemnatec.com) to capture an image at one time point and analyse the major morphological features, such as leaf area, rosette circumference and leaf area coverage (Fig. 2). The image capture unit allows for image recording in reproducible conditions once the light and camera parameters are set up
  3. analyse digital images. Digital images will be analysed based on object recognition and colour classification using the software, and morphological parameters will be compared.

 

The objective of Activity 1 is to compare the phenotype of the wild type versus mutant plants using traditional techniques (e.g., visual inspection, measurements of plant parts and leaf pigment observations) and image analyses.

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Figure 2. Non-invasive analyses of plant morphology

A) Images taken for five-week old wild type (WT) and mutant plants (M) and converted to false-colour using the LemnaTec system. B) Polar graphs representing five measured parameters of plant growth: relative growth rate (RGR), eccentricity, compactness, roundness and surface coverage. The data were normalised to the highest value in the series (axes scale is 0 to 1). C) Leaf area growth over time for four different genotypes; Columbia and C24 are wild types; alx8 and fry1–1 are mutant alleles (same gene) in the Columbia and C24 backgrounds, respectively.

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1.2) Materials

The materials required for this activity are:

  1. camera and tripod
  2. cold room or fridge to stratify the seeds and coordinate germination
  3. commercial seed-raising mix (mixes such as Debco work well); pasteurised soil is preferred to deter pest and pathogen growth
  4. slow-release fertiliser (e.g., Osmocote Exact Mini at 3 grams (g) of fertiliser per litre (l) of soil; mix after the soil has been pasteurised)
  5. filter paper
  6. forceps
  7. growth chamber
  8. magnifying glass
  9. mesh, non-slip PVC mesh (e.g., Matting Non Slip Magic Stop 90 centimetre (cm) Natural from Bunning’s) — optional
  10. plastic labels or coloured sticky tape to label individual pots
  11. plastic lids or cling film
  12. 48 pots per group (e.g., 63 millimetre (mm) Square Squat individual pots — Garden City Plastics Cat# P63SSQ)
  13. ruler
  14. seeds from wild type and mutant plants
  15. table outlining growth and development (Appendix C: Arabidopsis growth stages) and Excel spreadsheets (see Appendix B: What do I do with my data?)
  16. 31 x 44 cm plastic trays for storing your plants

1.3) Procedure

On the first day of the practical you will start from Step 4. Remember to always keep safe conduct in mind as you work (See Appendix A: General rules for safety and conduct).

Materials

Method

Soil, pots, fertiliser

  1. Mix Osmocote and soil at ratio of 3 g of Osmocote per l of soil. IMPORTANT:
  1. 4 l of soil are enough for a 24-pot tray
  2. add the soil to the pot and press gently
  3. a cement mixer can be used for larger volumes of soil (leftovers can be retained for Step 3).

Tray

  1. Place 24 pots per tray. TIP: it is a good practice to line the tray with a thin PVC mat to improve drainage.

Water

  1. Add 1 l of tap water to the tray and let it soak overnight. If you see the soil level has gone down overnight you can top it up with the soil left over from Step 1. The pots are now ready for sowing.

Labels

  1. Number labels from 1 to 48. Note the labels are indicated as ‘WT’ (wild type) or ‘M’ (mutant). It is best to have consecutive numbering to refer to individual plants (See ‘Labels’, Appendix B: What do I do with my data?)

Seeds, filter paper, trays, soil, Osmocote, pots, labels

  1. Sow seeds by placing them on a piece of filter paper and tapping them onto the soil. Be careful not to contaminate adjacent pots. Try to add no more than five–ten seeds per pot, and make sure they are not clustered in one spot. Sow seeds in half of the pots for each of the wild type and mutant lines. IMPORTANT: label each pot with a unique number (‘identifier’). You can link this number to a spreadsheet indicating genotype and condition.

 

  1. Randomise the location of the pots in the tray — i.e., don’t place all the mutants on one side and all the wild types on the other. Perhaps discuss with your group as to why we suggest this (make the most of your research efforts!).

Plastic lids or cling film, cold

  1. Cover the trays with a plastic lid or cling film and stratify at 4 ºC in the dark for 48 hours. This will help synchronise seed germination.

 

  1. Move trays to growth chamber or glasshouse. Add ~800 millilitres (ml) of tap water to each tray every other day (check with your instructor as this may be done for you).

 

  1. Remove plastic lids on day ten or 12.

Forceps

  1. Thin the plants to one–two per pot after the first two to four leaves are fully expanded (usually 14 days). IMPORTANT: don’t discard the plants you remove: you can use the thinned plants to examine below-ground phenotype — see Step 13.

Camera, ruler

  1. Observe the growth and development of the plants every week throughout the practical. Feel free to take photos or any other type of observation at any time. TIP: always place a ruler near the object when taking a photo to use as a scale bar.

 

  1. The Scanalyzer imaging schedule will depend on resources available and your goals.
  1. A single time point imaging of both wild type and mutant trays will allow you to compare morphological parameters.
  2. Multiple time points imaging (at least four times) between weeks three and five will allow you to calculate relative growth rates in addition to morphological traits.

Note that all plants will be imaged at the same time and that should give you enough statistical power to find significant differences.

 

  1. Analyses of roots. You can do this assessment on the seedlings you remove during thinning, as well as on the plants you harvest at the end. Periodic harvests along the way can also be informative, provided you have sufficient plants. To analyse the roots, VERY carefully remove them from the soil and gently wash under water. Arabidopsis roots are thin and fragile, so special care is required when cleaning them. Roots can be floated on a large Petri dish with water, scanned and analysed, as in Appendix E: Software tips.

 

Alternative procedures for morphological studies

  1. Trace individual leaves on a piece of paper. Cut the paper. Compare the weight of all pieces of paper per plant to a weighted piece of paper of known area. You will be able to estimate total leaf area by comparing the weight of the ‘paper’ leaves to that of the standard piece of paper.
  2. See Activity 9, Section 9.3.6 for more ideas about how to measure leaf area.

1.4) Expected outcomes

Record your observations about growth and development from at least five plants per genotype. Measure the same plants at each measurement date. Use your spreadsheet to identify which plant each measurement came from. The following lists some of the morphological parameters to observe and how to record them:

  1. number of leaves. Note that the first leaves, or cotyledons, are not considered to be ‘true’ leaves
  2. leaf shape, size and morphology; are leaves similar in form and shape?
  3. pigmentation; is there any difference in the colour of plant parts when you compare the wild type and mutant?
  4. rosette diameter; what plants have larger rosettes?
  5. stem (bolt) height, as measured from the base to the tip of the stem
  6. cauline leaves (number and length): the leaves produced in the stem, above the rosette
  7. flower/silique number and size
  8. the relative growth rate can be calculated RGR = (ln W2 – ln W1)/(t2–t1), where W1 and W2 are plant dry weights or leaf area at times t1 and t2 (Hoffmann and Poorter 2002).
  9. root traits, such as length, mass, colour and branching pattern
  10. digital images obtained with the Scanalyzer system, see Appendix E: Software tips (Arvidsson et al. 2011), for some options for data analyses. An example of data presentation is given in Fig. 2.
  11. graph and analyse your data, see Appendix B: What do I do with my data?

 

Note that the choice of parameters to measure is based on your preference and will depend on your observations. If you see something that suggests a difference between the wild type and mutant, take objective measurements of it. You are welcome to discuss with your instructors and peer mentors what parameters you would like to measure. Note that the plants will also be used for the other experimental assays, so keep your destructive assessments to a minimum.

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TIP: Quantifying qualitative traits

Often you will observe phenotypes that cannot easily be measured and quantified. For example, leaves might have slightly different colours or patches of coloured areas on them (Fig. 3A). There are different options to quantify such phenotypes:

  1. If you know what the pigments might be, you could try and extract them with organic solvents, and quantify the amounts per leaf using high-performance liquid chromatography (HPLC), as explained in Activity 5.
  2. Alternatively you may use qualitative assessment (Fig. 3B): Give each leaf a rank based on size and colour of the spot. Arrange leaves by rank, noting which one is wild type or mutant (it might be best to do this ‘blind’; i.e., work with a colleague who removes the labels as he or she hands you the pot, or write on the back of each leaf if it is wild type or mutant). You can use the rank order to do a rank sum test, such as the Mann-Whitney U test, to determine significant differences between wild type and mutant.

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Figure 3. Quantifying qualitative traits.

A) Schematic representation of five leaves from wild type and mutant plants with different colours or patches of coloured areas on them. B) The same leaves arranged on a relative rank based on size and colour of the spot. You can use the rank order to do a rank sum test, like the Mann-Whitney U test, to determine significant differences between wild type and mutant.

 


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