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Teaching the pressure-flow hypothesis of phloem transport in a problemsolving session

Paul Ciifford The Queens University of Belfast, UK

Problem solving is an ideai learning strategy, especially for topics that are perceived as difficult to teach. As an example, a format is described for a problem-solving session designed to help students understand the pressure-flow hypothesis of phloem transport in plants. Five key facts and their discussion can lead to the conclusion that a turgor-pressure difference along sieve tubes of the phloem drives a mass flow of sugar solution between source and sink. The given format is suitable for use in the sixth-form classroom when the mechanism of phloem transport Is usually first taught to students. Additional information is provided so that schoolteachers can lead a class discussion and stimulate student interest in the phloem as a long-distance transport system. Key words: Phloem transport; Pressure-flow hypothesis; Probiem solving

Introduction
It has always struck me as fascinating that so much in biology makes sense. The strands ofthe double helix of DNA must separate and synthesise a mirror image of themselves if two genetically identical cells are to be produced by mitosis. Likewise, a decreasing level of phytochrome in its far-red state would be expected to provide a timing mechanism for length of the dark period so that flower initiation can be triggered in angiosperms. Again, it has to be a pheromone that is produced by female silkworm moths if these can attract males that are several miles downwind. In other words, available facts fit with the way that a biological phenomenon is explained. Obviously they do, or the current hypothesis or accepted theory would be discarded. It follows that a well-designed, problemsolving session in which students are given key facts on a topic and asked to use these to come up with an explanation for how something works has every chance of success. The need to move teaching and learning processes towards a problem-based form has been recognised for both secondary and tertiary education (Phoenix, 2000]. This paper describes a problem-solving approach that can be used to teach the pressure-How hypothesis of phloem transport. An understanding of this model for long-distance sugar movement in plants is a requirement of most A-level syllabuses. Proposed by Munch in 1926, the hypothesis states that a mass flow of water and dissolved sugar is driven by a pressure difference through sieve tubes of the phloem from autotrophic organs called sources to heterotrophic organs called sinks. Although designed with the sixth-form (16- to 18-year olds) classroom in mind, the problem-solving session described here is suitable for
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any teaching situation in which the pressure-flow hypothesis is first introduced to students. Difficulties experienced by school teachers and misconceptions in A-level textbooks led to the choice of this topic for a problem-solving session (Chfford, 2002).

Method
Getting started

It is assumed that some knowledge of transport processes in plants has been delivered beforehand. Make sure that students understand how the plant body is organised in relation to transport by telling them about the symplast and apoplast. Terms like source and sink, phloem loading and unloading, osmosis, mass flow, turgor pressure and water potential should be familiar to the students. It is suggested that water uptake and xylem transport are covered before dealing with phloem transport. Students should not be forewarned of the problem-solving session or asked to undertake any preparation. This may tempt them to seek the answer from textbooks. Box 1 shows what is given to the students. There are five sets of facts. These have been selected as key information that could lead to the conclusion that there is a mass flow of sugar solution through sieve tubes driven by a turgor-pressure gradient. The problem has been stated in the form of a question. There is also a simplified diagram of the phloem transport system with a single arrow showing sugar loading at the source. Inclusion of this diagram can help students to envisage how a turgor-pressure gradient might be generated. The idea at this stage is to keep the problem as straightforward as possible. An opportunity to present extra facts will arise in a discussion involving the whole class.
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Box 1; Material given to students Problem solving - phloem transport Some facts about phloem transport are given below. These fit with the hypothesis currently in use to explain the mechanism by which sugars are moved in the phloem from source to sink. Look closely at these facts and use them to answer the question: "What is the likely mechanism that results in sugar movement in the phloem from source to sink?" You might find that this problem is tackled most easily by using a diagram. Make additions to the given diagram as ideas come to mind. A start has been made by inserting an arrow showing sugar loading at the source. The facts 1. Phloem consists of sieve tubes. These are formed from sieve elements arranged in files and elongated in the direction of transport. The sieve element is characterised by minimal cytoplasm, a broken-down tonoplast, no nucleus and sieve plates with unobstructed pores. P-protein may be present. 2. An average speed of phloem transport is 40 cm per hour. This is about 40 000 times faster than would be expected by diffusion alone. 3. Sugars are loaded at the source and unloaded at the sink. These processes require metabolic energy, but the transport mechanism itself is passive - metabolic energy is needed only to maintain the living condition of sieve tubes. 4. Sugar concentration in sieve tubes in relation to surrounding cells is very high - between 10-30% on a w/v basis or > 0.3 M. As a result, sieve tubes are under considerable turgor pressure. 5. 'Exotic' substances such as herbicides, dyes and radiotracers also move in sieve tubes. These move along with the sugars and follow the same source-sink distribution pattern. phloem
1

The class discussion

1 sink
sugar

A short [5 minute] preamble by the teacher is needed so that the students understand clearly the task in hand. This can take the form of reading through the instructions of the problem and answering any questions that come up at this stage. It is suggested that the class is split into 'buzz groups' of 3-4 students each. There is more chance of getting a response when you ask for comments from a group and have someone in that group act as a spokesperson than if you question individual students. Tell the students to look at each fact in turn and ask themselves what this fact tells them about the mechanism of phloem transport. They must then focus on the question as stated in the problem. It is suggested that 15-20 minutes will give students sufficient time to understand and tackle the problem.
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A discussion involving the whole class can now be led by the teacher. Take the students through each fact in turn. Encourage them to look for inter-relationships between these facts. Here are some hints on how to stimulate student interest and provide prompts as the class discussion continues. The problem-solving session can last for as little as 40 minutes, or be extended to two hours, depending on how much of the following information is covered. act 1 tells the students about the structure of sieve tubes. The given information relates to the so-called mature stage at which most sugar transport is believed to occur. An obvious conclusion is that movement of whatever moves in sieve tubes is relatively unrestricted. A mass flow of water and sugar is possible although this cannot be deduced from Fact 1 alone. The opportunity should not be missed to engage students in discussion of the fascinating nature of sieve tubes. Constituent sieve elements are alive despite the disintegration of both nucleus and tonoplast during development. A plasma membrane with some peripheral cytoplasm lines the cell wall. Embedded in this cytoplasm are a few mitochondria. The pores of sieve plates can be considered as enlarged plasmodesmata without cytoplasmic contents. Question the students on the significance of the lack of a nucleus. It means that sieve elements cannot synthesise proteins. This is consistent with the short life span of sieve tubes seen in most plants. The enucleate condition can be viewed as a means to maximise the volume of the cell for transport. A comparison can be made with the red blood cells of mammals. Another constituent of sieve tubes worthy of comment is Pprotein. There is nothing esoteric about this term - it refers simply to the proteinaceous material specific to phloem. The P-protein is dispersed evenly as a filamentous network in the lumen or 'empty space' of sieve elements. It is possible that P-protein serves as a clotting mechanism blocking the sieve pores when the phloem is injured. A chance to talk about the other cells that make up phloem tissue will present itself when dealing with Pact 3. You can tell students that the structure of sieve tubes - and hence the mechanism of transport - was once the subject of much debate. Some believed that P-protein was involved directly in the transport mechanism. One proposal had P-protein filaments localised in the pores of sieve plates with a mass flow of water and sugar driven by electro-osmosis. Another idea envisaged sugars being pumped in a peristaltic fashion inside tubules of P-protein whose walls consisted of contractile proteins. It was even thought that sugars could move in transcellular strands of streaming cytoplasm running through the pores of sieve plates. All these hypotheses had a direct requirement for metabolic energy and were made redundant in the mid 1980s once the information given in Pact 3 was accepted. Interestingly, P-protein is not found in sieve tubes of gymnosperms [and note that Pact 1 stated only that T-protein may be present'). Its absence in gymnosperms should have served as a warning to those who argued a case for active mechanisms involving P-protein. Fact 1 can usefully be linked with Fact 4 at this stage. Ask the students: "Why did microscopists find it difficult to determine the structure of sieve tubes?" The answer lies in their high turgor pressure. This means that sieve tubes are sensitive to handling so that their contents move when sectioned by conventional methods. The presence of P-protein filaments in sieve pores turned out to be an artefact caused by surge once pressure had been released.
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Phloem transport

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It took rapid fixation techniques coupled with electron microscopy before what was known as the 'open vs closed sieve pores' argument was finally resolved. Fact 2 gives the usual textbook speed for sugar movement in the phloem. A speed of 40 cm/hour is certainly impressive. At the cellular level, this is equivalent to 111 pm/sec. Tell the students that a typical sieve element is 200 pm long with a diameter of 20 pm. It follows that the length of a single sieve element is crossed in less than two seconds. In other words, sugars move at a speed of about five times the cell diameter for each second. Scale this up to a 5m wide river flowing at a comparable speed of 25 m/sec. You now have the kind of river that would raise the adrenalin levels of jet-boat enthusiastsl No wonder that the title of a recent review paper was: 'The phloem sieve element: a river runs through it'. The early investigators found a speed of 40 cm/hour difficult to reconcile with phloem tissue about the thickness of a postcard under the bark of a tall tree or the diameter of a human hair in a stalk leading to a large fruit. Experiments using aphids and '''C-Iabelled sugars provided the evidence that the phloem was the channel of transport. You might like to add that speed is not a good way to describe the performance of the phloem as a conducting system. As a measurement in cm/hour, it tells us nothing as to how much moves. In fact, speeds vary from 2 to 2000 cm/hour when determined by a variety of chemical, physical and tracer methods. There is no doubt that some of this variability has resulted from unreliable methodology. On the other hand, the possibility is raised that mechanisms in addition to pressure-flow operate to move sugars in the phloem. A better measurement is known as specific mass transfer. This is a rate expressed as the mass of sugars moved per unit area of conducting tissue per unit time. Values for specific mass transfer are about 5 g per cm^ of phloem per hour, but can be as high as 50 g per cm^ per hour at peak transport times in certain plants. Fact 2 also tells students that a speed of 40 cm/hour is about 40 000 times faster than simple diffusion. They should now appreciate that transport in sieve tubes is certainly efficient and involves a mechanism with a considerable energy input. The next fact deals with the thermodynamic vs metabolic relationships of phloem transport. Fact 3 gives the requirement for metabolic energy for the loading and unloading processes compared to the transport mechanism itself Tell students that two pathways exist for loading and unloading. The first - called the apoplastic pathway involves the cell wall and the plasma membrane while the second - called the symplastic pathway - involves cytoplasm and plasmodesmata. Link Fact 3 with Fact 4 by ensuring that students realise sugars are loaded against - and unloaded down - a concentration gradient. It follows that loading at the source has to be active, i.e. requires metabolic energy. In contrast, unloading at sinks could involve passive, in addition to active, transport processes. You can now talk about companion cells and transfer cells. Numerous plasmodesmata connecting the former to sieve elements would fit with a symplastic pathway. Cell-wall ingrowths that serve to increase the surface area ofthe plasma membrane are a feature of the latter. This points to an apoplastic pathway especially when associated with a lower plasmodesmatal frequency. A companion ceD is developmentally related to the sieve element being formed from the same mother cell. In contrast, a transfer cell is usually a modified phloem parenchyma cell. Companion
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cells and transfer cells are found at both source and sink. It is known that companion cells provide the life-support system for enucleate sieve elements in addition to any role that they have in loading/unloading. Studies have concentrated on understanding the apoplastic pathway. This is quite understandable since this pathway would be expected to offer the fine-tuning needed to regulate loading and unloading. The popular proposal to explain the loading step from apoplast into the sieve tube itself is that sugar and H"^ ions bind to carrier proteins in the plasma membrane and transport is driven by the electrochemical potential difference generated by H+ ions pumping in the outward direction. This is known as the sugar-H+ symport hypothesis of sugar loading. A similar mechanism in which carriers in the plasma membrane behave as antiporters might operate at the sink. Attention is now focused on the symplastic pathway. Some believe that sugars can even be loaded against a concentration gradient via this pathway. One proposal - known as the polymer trap model - involves synthesis in companion cells of largemolecule, sugar compounds with these only able to diffuse through the plasmodesmata adjoining sieve elements. Any symplastic pathway for unloading would be expected to involve diffusion and/or massflowthrough plasmodesmata with control dependent on their conductivity. The relative importance of these two pathways is still unresolved. Dyes that pass only through plasmodesmata and specific inhibitors of membrane transport have been useful probes for the determination of pathways. Current evidence suggests that the predominant pathway for loading is apoplastic in leaves of familiar crop plants such as sugar beet and wheat. The symplastic loading pathway may be a feature of less evolutionary-advanced plant families such as the cucurbits. Unloading is thought to be entirely symplastic in young, expanding leaves and symplastic with a final apoplastic step in such sinks as stem internodes and seeds. Seemingly, the pathway for loading is dependent on species, whereas that for unloading is dependent on sink type. Fact 3 eliminates active mechanisms that require a direct input of metabolic energy. Throw in the clue that all water movement in plants involves passive processes. Water flow in the xylem is driven by a gradient in negative pressure or tension brought about by transpiration. Students can now be encouraged to look for something similar to explain phloem transport. Faa 4 is a key fact. It holds true even for plants such as sugar cane and sugar beet where sugars are stored at the sink. This is also the first mention ofthe term 'turgor pressure'. Prompt students to think 'pressure in sieve tubes' by telling them about exudation from cut aphid stylets or cut inflorescence stalks of palm trees. Such exudation continues for hours or even days. This in itself provides indirect evidence for massflow.Get students to appreciate that turgor pressures of sieve tubes at both source and sink are much higher than those of adjoining cells. Give some values. A value of 1.76 MPa was obtained for sieve tubes of coconut palm using a pressure gauge attached to the cut inflorescence stalk. An even more ingenious technique was used to measure turgor pressure in sieve tubes of willow shoots. The length of a compressed air column in capillary tubing attached with superglue to an aphid stylet gave a value of around 1.0 MPa. In contrast, turgor pressures of leaf mesophyll cells range from 0.3 to 0.5 MPa. Link the high turgor pressures in sieve tubes back with Fact 1 by pointing out that densely packed, cellulose microfibrils
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in their cell walls prevent bursting by acting like hoops of a barrel. pressure. Both osmosis and mass flow are passive processes In addition, you can impress students by telling them that pres- requiring thermodynamic rather than metabolic energy for their sures inside any fully turgid plant cell are like those in the tyres operation. Sugar loading at the source can now be linked with water movement by osmosis into sieve tubes and a high turgor of racing bikes! At this stage, it could be useful to reinforce the concept of pressure. There is also the chance that students will wonder water potential in relation to the driving forces for water transport whether turgor pressures in sieve tubes are higher at the source by osmosis and mass flow. The familiar equation ^ = '^p + ^s than at the sink. Such a difference could cause a mass flow of shows that water potential of a plant cell, or any of its constituent water and sugar in sieve tubes with water entering at the source parts, is the sum of pressure potential and solute potential. and leaving at the sink. Pressure potential is negative if water is under tension and positive Fact 5 should really get the students to think that there must be if water is subjected to pressure [as it is for living plant cells, when a moving stream of both water and solutes. How else could a pressure potential is then numerically equal to turgor pressure]. In variety of substances not found naturally in the phloem be distribcontrast, solute potential due to dissolved solutes is always nega- uted from source to sink along with the sugars? You can add that tive. According to thermodynamic principles, water moves viruses are also found in the phloem. The fact that these can through an interface that solutes cannot cross by passive means enter and leave sieve tubes provides additional evidence for the (e.g. a differentially permeable membrane such as the plasma existence of a symplastic pathway for loading and unloading. membrane and for liquid to vapour transport during transpiration Dealing with Fact 5 gives you the excuse to enlarge on the from leaves] down a gradient of free energy from high to low solutes that are found naturally in phloem sap. At this stage, the water potential. The steeper the gradient, the greater is the transported carbohydrate has been referred to loosely as 'sugars'. It water movement. Osmosis can be defined as the movement of is known that sucrose is the main constituent of the sugars in the water across a differentially permeable membrane from low to majority of plants (and you can relate this back to Fact 3 by saying high solute concentration. For plant cells, this simple definition that the carrier proteins involved in sugar-H* symport bind only is not appropriate because rate and direction of water flux through with sucrose]. Sucrose may be up to 98% ofthe total carbohydrate, the plasma membrane are controlled by the water-potential gradient with small amounts of the reducing sugars glucose and fructose. with both pressure potential and solute potential acting as driving While this is the rule, there are some interesting exceptions. A forces. Massflowis the uni-directional movement of water as the group of sucrose derivatives called a-galactosides are the main result of a difference in pressure potential along the pathway. constituent of the transported carbohydrate in certain American Solutes would be expected to move simultaneously with the trees. Likewise, the sugar alcohols sorbitol and mannitol have water if there are no differentially permeable membranes to this role in apple and celery respectively. Other solutes found cross between cells as is the case with phloem and xylem. and transported in the phloem are nitrogenous compounds such It is now thought that massflowof water and solutes is possible as amino acids and amides. You can add inorganic ions to a list of through plasmodesmata (and this could be important for sugar solutes found in phloem sap. This will be puzzling for students. unloading via a symplastic pathway). Students should appreciate Potassium (K"^] ions are found in high concentrations in phloem sap despite the xylem being the channel of transport for this ion. that mass flow is independent ofa water-potential gradient because The presence of K+ ionsfitswith a version of the sugar-H+ symport the only component of water potential that matters is pressure potential. Solute potential cannot act as a driving force for water hypothesis in which outward H* pumping is coupled with K* movement although it must remain in the '^ = '^p + '^'s equation. pumping in the opposite direction. Inorganic phosphate (Pi] In fact, solute potential is one component of water potential ions are present in smaller amounts, nitrate (NO3 ]ions are comthat can never be removed for the purpose of calculating water pletely absent and there are traces of the other inorganic ions. potential. This is obvious since the water potential of a sucrose Hormones such as abscisic acid (ABA] and gibberellic acid (GA3] are also found in phloem sap. solution in a beaker has to be its solute potential. In contrast, a The most interesting constituent of phloem sap to come to light component of water potential like pressure potential can sometimes be ignored (e.g. when its value is 0, as it would be for a in recent years has been mRNAs. These originate in companion cells and find their way into sieve elements via plasmodesmata. sucrose solution in a beaker]. There is some extra information that can be added to this The possibility is raised that mRNAs of source origin act as a explanation. Note that 'movement' rather than 'diffusion' was means of source-sink communication by determining protein used when defining osmosis. It is known that water crosses the synthesis in sinks. Finally, you can add that phloem sap is alkaline plasma membrane by a combination of diffusion through the with a pH of around 8.0. Thisfitswith outward pumping of H+ ions lipid bilayer and mass flow through protein channels called as envisaged by the sugar-H* symport hypothesis of sugar loading. aquaporins. Such mass flow is termed correctly since it is due to a hydrostatic pressure gradient created by an uneven concentration Discussion of water molecules along the length of the aquaporin. For this This author does not claim that the five sets of facts are necessarily in the best order. Facts 3 and 4 are possible candidates for massflow,solutes cannot move with the water because aquaporins are water-selective. Students will have to take on board that mass reversal. The given information could also be modified. For flow can occur across the plasma membrane as well as through example, the mention of P-protein in Fact 1 could be considered plasmodesmata, the xylem and the phloeml It is thought that as an optional extra. Likewise, you might like to let the students aquaporin availability controls and fine-tunes osmotic water reach the conclusion that sieve tubes are under high turgor pressure rather than give this in Fact 4. flux through the plasma membrane. Extra facts might need to be given. For example, it could be Taken together. Facts 3 and 4 have the students looking for a difficult for students to imagine a turgor-pressure gradient driving passive mechanism operating in sieve tubes with high turgor
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mass flow unless they are told that different sugar concentrations are meant to exist in sieve tubes at source and sink i.e. high turgor pressure is associated with high sugar concentration, and vice versa. Some facts should be held back until a later stage. For example, there is no point in discussing the xylem as a return pathway for water movement until students have reached the conclusion that water is moving in sieve tuhes. Be aware that the xylem can only be the return pathway for water movement in the case of roots. For shoot sinks, the water presumably enters the xylem and is then lost in transpiration. The problem-solving session starts to get more complicated if students ask what makes water leave sieve tubes at the sink. A suitable answer to this and other questions can be found in the earlier paper on this topic [Clifford, 2002). Hopefully, you wiU end up saying to students: "Congratulations. You have just come up with the generally accepted explanation for sugar transport in the phloem - the pressure-flow hypothesis!" If not, resort to the diagram in Box 1 and use this to explain how sugars could move from source to sink. Start with sugar loading. This lowers the water potential of sieve tubes at the source. Water influx by osmosis through the plasma membrane of sieve tubes then follows. The resultant increase in turgor pressure drives a mass flow of water and sugars through sieve tubes. A lower turgor pressure is maintained in sieve tubes at the sink as a result of sugar unloading. There must also be a downward gradient in water potential across the plasma membrane of sieve tubes at the sink so that water can leave the phloem. Alternatively, use the working laboratory model described in most textbooks. This consists of two osmometers connected by glass tubing and sited in a water bath. Initially, the whole system contains only water. Sugar is introduced by some means into one of the osmometers. The explanation proceeds along similar lines except that you should refer to hydrostatic rather than turgor pressure since living cells are not involved. It is an increase in hydrostatic pressure in the second osmometer that leads to water efflux. Massflowwill stop when sugar concentrations in the two osmometers are the same. The model is applicable to the plant situation if sugars are loaded at the source and unloaded at the sink. There is a problem with any step-by-step explanation. Students might fail to appreciate the dynamic nature of mass flow in sieve tubes. For example, saying that 'sugar loading lowers water potential of sieve tubes at the source' is simplistic when sugar loading is a continuous process that maintains the water-potential gradient needed for osmotic water influx. Nonetheless, a stepby-step explanation does provide a simple description of the pressure-flow hypothesis in a few, carefully worded sentences.
Current status of ttie pressure-flow hypothesis

Phloem physiologists believe that the original pressure-flow hypothesis might be in need of some modiflcation. One of its assumptions is that the turgor-pressure gradient is produced by different sugar concentrations in sieve tubes at source and sink. These have been difficult to demonstrate at least in small herbaceous plants. For this situation, it has been suggested that the osmotic gradient along sieve tubes is determined more by potassium (K+] ions than by sugars. Another assumption is that water leaves sieve tubes at the sink entirely through the plasma membrane. There is now evidence that some water exits as a mass flow through plasmodesmata with unusually large-size exclusion limits. The low turgor pressure need for sink control of mass flow could even be located in non-vascular cells. You could complicate the story by teUing students that experimental evidence for water movement in sieve tubes is lacking. Attempts using -^H-labelled water have failed to show that water moves along with the solutes. This is a limitation of the pressureflow hypothesis. The counter-criticism is that water movement cannot be demonstrated using a radiotracer because water readily leaks out of sieve tubes. Water is moving, but does so as through a hose pipe with numerous holes. This is one reason why the pressure-flow hypothesis remains a hypothesis rather than established theory, despite its proposal by Miinch back in 1926.

Concluding remarks
Problem solving as an experiential learning strategy is well suited for teaching at A-level when many topics are taught for the first time. Just assemble some key facts and construct a suitable question. Do this especially for those topics in plant and animal biology that are perceived as difficult to teach. Students will develop problem-solving skills as well as gaining a better understanding of the topic in question. Misconceptions should be reduced so that an understanding can be built on at university level. The take-home message of this paper is that teaching in problemsolving sessions will be more effective and much more fun than using conventional teaching methods.

Acknowledgements
I thank John Patrick for his valuable comments especially in ensuring that this paper provides a sound understanding of the pressure-flow hypothesis. Thanks also to Robin Govier and Hugh Fletcher for constructive criticism of an earlier version of this paper.

References
Clifford P E (2002) The pressure-flow hypothesis of phloem transport: misconceptions in the A-level textbooks. Joumal of Biological Education, 36, 110-112. Phoenix D A [2000) Looking towards reform - the student focus.
Joumal of Biological Education, 34, 171.

The pressure-flow hypothesis is widely accepted as the mechanism of phloem transport. Those in favour of this model estimate the pressure difference needed for mass flow of water and solutes through sieve tubes of known resistence using the Poiseuille Equation. This takes into account such parameters as speed of transport, viscosity of the moving solution, dimensions of sieve tubes (including the size of pores in sieve plates), and frequency of sieve plates. A calculation for sieve tubes of pumpkin has given a value of 0.04 MPa per metre of sieve tube. Such turgoripressure gradients are considered as easily available.

Paul Clifford is an Honorary Senior Teaching Fellow in the School of Biology and Biochemistry, The Queen's University of Belfast, Medical Biology Centre, 97 Lisbum Road, Belfast BT9 7BL, Northern Ireland. Teh +44 (0) 28 9027 2124; Fax: +44 (0) 28 9097 5877; Email:p.cUfford@qub.ac.uk

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