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

General information about the Plant Detectives Project

Introduction

The rate at which we must increase food production needs to grow with every increase in world population growth (United Nations Food and Agriculture Organisation). This scenario is further complicated by the uncertainty of climate change and biodiversity loss. Meeting increased food demand under ever limiting conditions while protecting natural resources will be a major challenge. The next generation of plant scientists will need creativity backed by high quality knowledge and investigative skills if they are to tackle this challenge.

We believe that a research-led approach provides an excellent theoretical and practical understanding of the integral links between genes, cells, whole plants and the environment that will be required for solving some of these future challenges. Over the next weeks, you will put into practice your newly acquired theoretical knowledge as you apply cutting-edge laboratory techniques to the “puzzle” of pinpointing genetic mutations in plants. In this process, you will get hands-on experience, test essential concepts in plant biology and explore, investigate and assess the effects of genetic mutations on plant form and function. We hope this will motivate and inspire you to learn more about plant biology, and help you develop the problem-solving curiosity, questioning and resourcefulness that one needs to be an effective researcher.

Mutants play a major role in understanding how plants work

The isolation and identification of mutant plants has played a major role in understanding plant physiology and is a regular screening procedure for genetic studies. Although mutations can arise naturally in all species, it is relatively easy to induce changes in a plant’s genetic makeup by treatment with chemicals. Observation of the resulting external and internal features, physiological changes and molecular profiles (i.e., transcripts, proteins, and metabolites) caused by a mutation can provide important information about the role of the gene that is affected.

This laboratory manual provides laboratory techniques for the study of plant morphology and physiology of the model organism thale cress (Arabidopsis thaliana, hereafter Arabidopsis). The set of activities used here is not, however, limited in application to this well know model system; they can be applied to any species. It is hoped that the laboratory exercises presented, along with the appropriate theoretical information, will provide you with fundamental knowledge of the links between genes, plant form and function.

Image

Figure 1. Arabidopsis accessions, distribution and morphology.

A) Top and side views, and individual leaf number 6 of different Arabidopsis accessions after 4 weeks. B) Distribution map of known accessions. Red dots represent likely introductions. C) Schematic representation of Arabidopsis organs. D) Example of Arabidopsis mutant. A, B, adapted from (Weigel 2012).

Why did we choose Arabidopsis?

The flowering plant Arabidopsis is a member of the mustard family (Brassicaceae) native to the Northern hemisphere (Fig. 1A-B). Arabidopsis has been extensively used as a model plant for genetic and physiological studies for half a century (Meinke et al. 1998) and has played a major role in our understanding of plant biology (Lavagi et al. 2012). Arabidopsissmall diploid genome (125 Mb) was the first plant genome fully sequenced (Initiative 2000). Arabidopsis has five chromosomes and a short life cycle (six weeks from germination to mature seed) and its small physical size makes it particularly amenable for laboratory research. There are over 7000 natural accessions, or “ecotypes”, of Arabidopsis, each with distinctive genetic make ups providing adaptation to specific environmental conditions (Weigel 2012). There is also a plethora of mutant lines of Arabidopsis, some natural and some generated in the lab. Both ecotypes and large mutant populations are available through stock centres, like Nottingham Arabidopsis Stock Centre (NASC) and the Arabidopsis Biological Resource Centre (ABRC) in Ohio.

One way of generating Arabidopsis mutants is by treating seeds with chemicals, such as ethyl methanolsulfonate (EMS), or radiation to create lesions in the gene sequence. Seeds are sown and the resulting M1 generation grown to flowering stage. At this stage, it is expected that any particular lesion in a DNA locus would have occurred in only one of the five chromosomes. M1 individuals are “heterozygous” for that particular mutant allele. The M1 plants produce flowers, and because Arabidopsis plants self-pollinate (meaning that the pollen and ovule come from the same individual) they will go on to produce seeds. Some M1 plants harbouring dominant mutations can present in a different phenotype (that is, observable characteristics). To detect the effect of recessive mutations, the self pollinated seeds from the M1 plants are sown, giving rise to a homozygous M2 generation. Seeds from the M2 generation are then collected and, either the seeds or the resulting plants can be screened for recessive mutations by comparing specific traits to the wild type, such as morphology, pigmentation, etc. (Fig. 1D). It is expected that 1 out 4 plants will be homozygous for specific mutant alleles in the M2 plants. Because random mutations can occur in other loci, the isolated mutant plants are usually backcrossed to the parents in order to “clean” any spurious secondary mutations.

Working with Arabidopsis

Arabidopsis plants are commonly grown either on plates containing nutrient medium and agar, or in soil. Soil can be supplemented with slow release fertilizers, such as Osmocote Mini Exact, or Hoagland’s solution. Because of their small size and short life cycle, they can be grown in high density in either growth chambers or greenhouses. A major factor in plant morphology and life cycle is the photoperiod (i.e. hours with light within a 24 hour period). Plants grown at shorter photoperiods (e.g. 8 hours light) grow slower and take longer to flower than plants grown at longer hours in the light (e.g., 16 hours). Watering by subirrigation is preferred and the amount of water and frequency of watering depends on the specific pot size and number of plants. TIP: It usually takes ~800 ml of water per 31 x 44 cm tray every 2-3 days to keep plants growing happily. It is very important not to overwater: if the top soil is wet, do not provide additional water.

Objectives of the Plant Detectives Project

The major objective of the Plant Detectives Project is to demonstrate the links between variation at the genetic, molecular and phenotypic level in different types of environments. In this case, our experimental setting will be a glasshouse, or growth chamber, and you will subject the plants to a range of growth conditions (e.g. drought) to assess the impact of genetic changes on the plant.

In this project you are a “plant detective” who needs to use the scientific method to discover genetic changes manifested by differential responses between the mutant and the wild type. For this, you will be given seeds from wild type and mutant Arabidopsis. Although unknown to you, the mutant will be one that has already been published1. Only one member of the teaching team will know which mutants are being used or which students have them. Your demonstrators, peer mentors and academics will not know the identity. In each practical session, you will perform a set of experiments to identify the phenotypic effects of the mutation on the plant morphology, anatomy, physiology and biochemistry. It is expected that at the end of the class you will be able to infer your “unknown” mutant by comparing your findings with those in the literature.

By doing this, you will become familiar with some of the most widely used and important techniques in contemporary plant biology around the world. You will also learn about plant physiology and morphology by monitoring the changes in the wild type and mutant plants in response to different environments. These techniques are regularly used by plant biologists (including geneticists, ecologists, physiologists, biochemists, developmental and molecular biologists). They are the current standard, and you will find they have very broad application.

To meet the above objective you will work in groups and will investigate your unknown Arabidopsis mutant by performing the following assays:

  1. Calculating seed germination rates, root growth and development
  2. Observing internal and external phenotypic features throughout germination and development
  3. Analysing pigment composition of plant parts
  4. Measuring the gas exchange characteristics (photosynthesis and transpiration)
  5. Assessing effects of water availability (and potentially other growth treatments of your choosing) on the above plant characteristics

 

Before the practical component

You will need to complete a brief on-line quiz before each laboratory session. This is to encourage reading of the appropriate protocol before you come to the lab so that you know in advance what is expected of you and what the day’s objectives are. You will receive 2% for completing each quiz. Quizzes will be available online for 1 week before the laboratory session, and will close 15 minutes before the session begins.

Please note, these Quizzes are largely going to run on an honour system. You are welcome to discuss the answers among yourselves before completing the quiz. We will not give the 2% to any students whose answers are identical or for nonsense answers.

The questions on the quiz will be the same each week and are listed below. Read your lab protocols and your class notes in order to complete the quiz.

1. Describe and interpret your results from the previous lab session (from session 2 onwards)

Compile all the data (and share between your group members) and describe the results that you’ve obtained from the previous experiment. The results need to be presented in a conclusive form. Give us numbers or percentage, for example, “The germination rate for the mutant is 54%”. We don’t want vague results like “Some of the mutants germinated”. The results will then need to be interpreted accordingly. You may refer to your lab manual, lecture notes or additional journals to interpret your results. See Appendix B for tips on how to handle your data. This will also help you with the write up of your final report.

2. Describe this week’s experiment

Read your lab manual and understand the experiment that you’ll be doing for this week. Understand the rationale behind the experiments and summarize about what you’ll be doing in the lab for this week’s prac.

3. Explain the objectives of this week’s experiment

These are described in each activity, but we’d like you to put them in your own words. Explain what you understand about the objectives of the experiment. Dot points are fine but be specific.

4. Explain one of the techniques for this week’s experiment in more detail

Some of the experiments will require you to use more than one technique. You may choose any of the techniques for this week’s prac and explain what you understand about the technique, including the purpose and the applicability of the technique in regard to your experiment.

5. What is your hypothesis for this week’s experiment in regard to your mutant?

Based on your growing understanding of your mutant (e.g. results from previous week’s experiments) and the objectives and experimental techniques that you’ll be doing this week, try to state a hypothesis regarding your mutant. Make your hypothesis precise and descriptive: not simply ‘I hypothesize that x will be different from y’, but ‘I hypothesize that x will be greater than y because . . . ’

During the laboratory session: Take notes on the practical material in your laboratory notebook. Number the notebook pages and record the date during each class. We expect you to make notes of what you did, record observations and describe results, draw pictures of anatomical structures etc. We will provide questions to guide your thinking. Your entries will serve as notes to yourself. If you take your laboratory journal seriously you will end up with an excellent study tool and reference for future years. Record what you learned and keep notes on questions that you have about what you have done. Bring your notebook to every laboratory session

Expected outcomes

The Plant Detectives project has no “right or wrong” outcome. Instead, the goal is to use this model system to give you a chance to explore ‘doing science’. You will have the opportunity to make your own links between gene (or genes) and phenotype. Based on your interpretation of results and literature research you will develop a solid understanding of modern practice and the growing connections among the fields of genetics, physiology, biochemistry and plant ecology.

Assessment

The goal of this project is to identify the mutant, i.e. identify the function of the mutated gene, or at least hypothesise what it could be, and to describe the effects of the mutation on the phenotype. This will be based on results of the lab work and literature research. The final mark is not based on whether or not the right answer (i.e., the gene) is identified, but on the approach taken, and the interpretation of the results used in identifying the likely suspects.

Assessment will consist of a write-up in the format of a scientific paper and a symposium presentation to the class. The class presentations in particular are a great opportunity to share with your peers the outcome of you research. Since each group is investigating a different mutant, the symposium will showcase a broad range of gene to environment connections.

Working groups and peer mentors

Students will be organized in “Teams” of two to five members (depending upon class size) to perform experiments and analyse the results during the eight weeks of intensive laboratory-based research. The laboratory session starts with “Discussion groups”, made up with one member from each team. The aim of the Discussion Groups is for students to share their findings across teams and compare results from the previous sessions, reflecting the way in which scientists collaborate.

Instead of or in addition to classic demonstrators, we favour the use of paid “Peer Mentors”, usually enthusiastic students from the previous year’s cohort identified through a selection process. Peer Mentors facilitate the cross-team discussions and work closely with laboratory groups to bridge the gap between researcher and student. In this process, the Peer Mentors also benefit, as they are taught by the teaching team how to facilitate critical thinking rather than reveal ‘answers’.

Experimental Outline

A clear outline of the experimental investigation to be performed is critical for the success of the “Plant Detective” activities. Your instructor will provide you with one of these. The actual order of the activities in this manual will be customized by your instructor to suit your course circumstances and will include dates and type of activities, necessary preparation, the teaching staff who will be present (Peer Mentors and instructors). Description of experimental procedures and age of the material are also important for the planning and execution of the experiments. Some of the experiments may differ in timing depending upon how your mutant plants develop. A second sowing, usually in week 2 (under supervision), is highly advisable because it gives you hands-on experience with plant growth and care and as well as providing backup material for extra experiments. For more information on Arabidopsis development see Appendix D. The experimental outline will help you gaining a broader outlook of the project.


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