Recent research has identified a karrikin (a butenolide derative) known as 3-methyl-2H-furo[2,3-c]pyran-2-one, formed from burning cellulose, that stimulates seed germination. Here, I present ideas on how to investigate the influence of karrikins on seed germination in the laboratory.

So often, school laboratory work does not promote guided inquiries or investigations, and Herron (2009) reminds us that many of these laboratory “investigations” merely serve as cookbook or verification activities. Hailman (1975) argued more than three decades ago that the approach to the “scientific method” in schools was often just as detached from how an Einstein functions as the color-by-numbers sets are removed from Michelangelo’s painting technique. We need to ask ourselves whether laboratory investigations today give students true insights into the nature of science and recent developments in science. In a guest editorial in ABT (August 2010, pp. 334–336), Lawson eloquently formulated a number of questions to guide students during investigations. Using some of these questions as schemata, this How-To-Do-It allows students to explore the influence of an active butenolide, 3-methyl-2H-furo[2, 3-c]pyran-2-one (Figure 1), on seed germination. The role of smoke in seed germination, especially in biomes like the chaparral in California, is currently receiving a lot of attention. This research can obviously have significant economic implications in the future. This activity might especially appeal to teachers in Advanced Placement programs.

Figure 1.

Chemical structure of 3-methyl-2H-furo[2,3-c]pyran-2-one.

Figure 1.

Chemical structure of 3-methyl-2H-furo[2,3-c]pyran-2-one.

Methods

I suggest that you allow students to work in groups of four. Provide them with the information in the “Karrikins & Seed Germination” section below, and instruct them to read more on the topic, discuss the possible influence of karrikins on seed germination, formulate a hypothesis, plan an investigation, record their results, and design a poster communicating their findings. You could perhaps provide students with key words (e.g., karrikins, butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one, smoke, and seed germination) and ask them to use a search engine to find relevant information on the Internet. The following questions are useful to guide students during their investigations (based on Lawson, 2010):

  • What, according to the literature you consulted, is the influence of karrikins on seed germination?

  • What is puzzling about what you read?

  • What are some possible explanations (hypotheses)?

  • How might these explanations be tested? (Design an experiment to test your hypothesis.)

  • Which factors might negatively affect your experimental design?

  • What are the expected/predicted results of the planned test(s)?

  • Following your test, what are your observed results?

  • How do your observed and expected results compare?

  • Design a poster, in which you communicate your findings.

One of the factors that will probably negatively influence the experiment is fungal growth. If you want your students to experience something of the “messiness” of scientific research, you should perhaps not flag this. However, if time is of the essence and you do not want students to start afresh after a few days, because of seed contamination, you could suggest to students that they exchange the wet cotton wool every second day or so, or use a fungicide. However, the latter may also inhibit some plant species’ germination, and some seeds may be very sensitive to fungicides.

Briquettes are a good source of fuel for a fire, and also safer than wood in the classroom. The briquettes sold commercially for cooking food include ingredients such as wood charcoal, sawdust, straw, and sunflower husks, which all contain cellulose, as well as other materials like wax (to facilitate ignition) and starch (a binder) (Grover & Mishra, 1996). Because of the cellulose present in the briquettes, butenolides (karrikins) are released when they burn.

Two or three briquettes, in a big tin, are sufficient to provide enough smoke for the investigation. Although I made use of bean seed, you may want to have a more authentic investigation, in which students use species that occur in more Mediterranean climates like the chaparral. You could consider the seed of plants such as Ceanothus species (that require intense heat and smoke for germination) or several pine species such as the knobcone pine, Bishop pine, or Sargent cypress (see Box 1 for more information).

Box 1: Further Reading & Safety Precautions

In addition to the references I consulted (see References), the following articles and contact details may be useful:

Flematti, G.R., Ghisalberti, E.L., Dixon, K.W. & Trengove, R.D. (2004). A compound from smoke that promotes seed germination. Science, 305, 977.

Light, M.E., Burger, B.V., Staerk, D., Kohout, L. & Van Staden, J. (2010). Butenolides from plant-derived smoke: natural plant-growth regulators with antagonistic actions on seed germination. Journal of Natural Products, 73, 267–269.

More information on the plants of the chaparral can be obtained from the CDF Headquarters, PO Box 944246, Sacramento, CA 94244-2460. They can be contacted at (916) 653–5123 or (916) 653–7958.

Safety Issues

Briquettes are a safer option to use than wood. Do the activity outside the classroom in a well-ventilated area. I do not recommend the use of fungicide, but if students use it, make sure that they apply it in your presence.

Karrikins & Seed Germination

Provide your students with a factsheet that highlights the following information. It is widely recognized by plant physiologists that forest fires have the ability to stimulate the germination of seeds. In the more Mediterranean biomes (e.g., the chaparral in California, and the fynbos vegetation in South Africa), fire is essential for some plant species’ seeds to germinate. Research has shown that chemicals in the smoke are the cause of such seed germination. Smoke is a complex mixture of thousands of different compounds, but Gavin Flematti and his coworkers (2004; see Box 1) discovered the chemical responsible for this effect. Butenolide derivatives known as karrikins are plant growth regulators found in the smoke of burning plant material. These karrikins are formed from burning cellulose, the molecule that makes up the cell walls in plants. Recently, a highly active butenolide, 3-methyl- 2H-furo[2,3-c]pyran-2-one, also known as KAR1, was isolated from smoke as a stable and volatile growth stimulant (Van Staden, 2010). Research has indicated that karrikins make seeds more sensitive to sunlight, so that plants emerge with less exposure. Studies also seem to suggest that exposure to karrikins accelerates seedling growth and resistance to stresses (such as drought) in a large number of plants.

The term karrikin is derived from karrik, which means “smoke” in the language of the Noongar people, the native inhabitants of Western Australia. This provides the opportunity to introduce indigenous knowledge in the life sciences classroom. Prior to the arrival of Europeans in Australia, tens of thousands of Noongar people lived in southwestern Australia. Colonization by the British resulted in violence, which took a heavy toll on the population, today estimated to number 21,000–28,000. The Noongar were environmentally literate people who divided the year into six distinct seasons that determined their migration, hunting, and agricultural activities (Green, 1984; http://noongar.org.au/).

Results

Students should design their own experiment to test their hypotheses. They might decide on an experiment to test for the influence of karrikins on seed germination, or to determine whether seedlings exposed to karrikins are more resistant to stress (such as drought). The results of my own investigation are presented below (Table 1).

Table 1.

Mass of (A) control seeds, not treated with smoke, and (B) seeds exposed to smoke for 15 minutes, for beans and squash.

Bean seeds (10 seeds in each of the control and experiment)
Day (A) Control (B) Smoke treatment (Experiment) 
1 4.5812 g 4.5723 g 
6 13.8889 g (+9.3) 18.0488 g (+13.47) 
9 15.8592 g (+11.28) 24.7228 g (+20.15) 
Squash seeds (10 seeds in each of the control and experiment) 
1 1.0466 g 1.1251 g 
6 1.7052 g (+0.6586) 1.8906 g (+0.7655) 
9 1.9301 g (+0.8835) 2.2299 g (+1.1048) 
Bean seeds (10 seeds in each of the control and experiment)
Day (A) Control (B) Smoke treatment (Experiment) 
1 4.5812 g 4.5723 g 
6 13.8889 g (+9.3) 18.0488 g (+13.47) 
9 15.8592 g (+11.28) 24.7228 g (+20.15) 
Squash seeds (10 seeds in each of the control and experiment) 
1 1.0466 g 1.1251 g 
6 1.7052 g (+0.6586) 1.8906 g (+0.7655) 
9 1.9301 g (+0.8835) 2.2299 g (+1.1048) 

Hypothesis

Exposure to the butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one, will stimulate seed germination.

Experimental Design

I need to admit that my first, original experimental design was flawed, although it produced wonderful results! I acknowledge the advice of Professor William Leonard in this regard. Lazy housewife beans (Phaseolus vulgaris) were used in the original investigation. I used burning cigarettes (which, of course, contain cellulose) as a source of smoke. Professor Leonard highlighted two problems with this design: first of all, cigarettes are not allowed in U.S. schools. Secondly, another variable is brought into the equation: nicotine. Some students might design experiments in which they plan on using cigarettes, and this can be a good discussion point: How does one conclude whether better germination is attributable to the smoke treatment or, perhaps, to the effect of the nicotine?

The following methodology used during my second attempt provided very good results and might be a good design to use in the classroom. The beans in the control were not exposed to smoke; the beans in the experimental groups were exposed to smoke (from burning charcoal briquettes) for 15 minutes. In this investigation I placed burning briquettes in a large empty coffee tin (Figure 2). Cover the opening of the tin with either a fine net (gauze) or filter paper, and place the seeds on top. (At this stage, the seeds should not yet have been exposed to water.) After the experimental seeds were exposed to the smoke, both the control and experimental seeds were placed between two wet pieces of cotton wool and left in a cool place (but exposed to daylight). Research has indicated that karrikins make seeds more sensitive to sunlight. (An alternative investigation might be to expose both the control and the experimental seeds to smoke, but to place the control seeds in a dark cupboard and expose the seeds in the experimental group to light.) It is essential to change the cotton wool every second day, or alternatively to use a fungicide, to prevent fungal growth. Seeds should be examined every day. Once germination takes place, students should measure the length of the shoots or determine the mass of the germinating seeds (see Figure 3). There are a number of issues that you should highlight, including imbibition (the absorption of water by the dry seed) and environmental factors that affect germination (e.g., temperature and light). It is recommended that students also illustrate their findings graphically (see Figure 7).

Figure 2.

(A) Burning charcoal can be put in a large tin. (B) Exposing the experimental seeds to smoke (cover the tin with gauze). (C) Different species were used, and beans provided the best results.

Figure 2.

(A) Burning charcoal can be put in a large tin. (B) Exposing the experimental seeds to smoke (cover the tin with gauze). (C) Different species were used, and beans provided the best results.

Figure 3.

(A) The control and experimental bean seeds on days 1, 6, and 9. (B) The control and experimental squash seeds on days 1, 4, 6, and 9.

Figure 3.

(A) The control and experimental bean seeds on days 1, 6, and 9. (B) The control and experimental squash seeds on days 1, 4, 6, and 9.

Figure 7.

(A) The mass of control and experimental bean seeds over a period of 9 days. (B) The mass of control and experimental squash seeds over a period of 9 days. Students can be requested to draw graphs of their experimental findings.

Figure 7.

(A) The mass of control and experimental bean seeds over a period of 9 days. (B) The mass of control and experimental squash seeds over a period of 9 days. Students can be requested to draw graphs of their experimental findings.

I made use of different types of seed – pumpkin, squash, beetroot, and beans. Bean seed consistently provides the best results, and I recommend this for classroom use, unless you want to use plants that occur in the chaparral to contextualize this investigation in terms of the role of fire in some biomes. However, it might not be easy to obtain seeds like Ceanothus species, and having done three trials with bean seeds, I know that a teacher can expect good results with the latter. If you use bean seeds, just contextualize the activity by stating that in certain biomes fire (and smoke) is essential for germination of some species.

Findings

After 72 hours of imbibition, a much higher percentage of smoke-treated bean seeds exhibited radical emergence, compared with the control (this is true of both variants of beans used – lazy housewife and bushbean). This is in line with the findings of other research, such as that of Jain and Van Staden (2007). Seeds exposed to smoke germinated better than seeds in the control (not exposed to smoke). In the case of squash and beetroot seeds, it took longer for radical emergence and germination (see Figure 6). Given the limited time allowed within a full biology curriculum, I therefore recommend the use of seeds such as beans that germinate quickly.

As can be seen in Figures 4, 5 and 6, seeds exposed to smoke for 15 minutes germinated the best – especially for the two varieties of bean seeds. Results were less dramatic in the case of squash.

Figure 4.

Bushbean seeds compared on day 9.

Figure 4.

Bushbean seeds compared on day 9.

Figure 5.

Lazy housewife beans compared on day 9.

Figure 5.

Lazy housewife beans compared on day 9.

Figure 6.

Squash seeds compared on day 9.

Figure 6.

Squash seeds compared on day 9.

Conclusion

It is clear that exposure to smoke (3-methyl-2H-furo[2,3-c]pyran-2- one) resulted in better germination. Have a class debate on whether these findings have any economic implications.

Suggestions for Further Investigations

As suggested above, several hypotheses can be formulated, and students could investigate the following:

  • Does the time of exposure to smoke (e.g., 15, 45, and 75 minutes) have an influence on germination? Is there a direct proportional relationship between germination and the time of exposure?

  • Is there a difference in the germination of smoke-treated seeds in the absence (control) and presence (experiment) of light?

  • Are seedlings exposed to smoke (experiment) better adapted to conditions of drought than seedlings that were not treated with smoke (control)? Germinate seeds according to the procedure described (and initially both control seeds and experimental seeds are put between two wet sheets of cotton wool). After the seeds germinate (day 5), they should first be weighed, placed between two dry sheets of cotton wool, and then inspected twice daily. Is there any evidence that the seeds exposed to smoke are more tolerant of drought conditions?

  • Do all seeds germinate better when exposed to 3-methyl-2H-furo[2,3-c]pyran-2-one? Students could replicate the experiment for different seeds. (As can be seen, the germination of squash seeds was less affected by exposure to smoke than that of beans. Beetroot showed hardly any response in my investigation.)

Students (in their groups) could be asked to design posters to communicate their findings.

References

Flematti, G.R., Goddard-Borger, E.D., Merritt, D.J., Ghisalberti, E.L., Dixon, K.W. & Trengove, R.D. (2007). Preparation of 2H-furo[2,3-c]pyran-2-one derivatives and evaluation of their germination-promoting activity. Journal of Agricultural and Food Chemistry, 55, 2189–2194.
Green, N. (1984). Broken Spears: Aborigines and Europeans in the Southwest of Australia. Perth, Australia: Focus Education Services.
Grover, P.D. & Mishra, S.K. (1996). Biomass Briquetting: Technology and Practices. Bangkok, Thailand: FAO.
Hailman, J.P. (1975). The scientific method: modus operandi or Supreme Court? American Biology Teacher, 37, 309–310.
Herron, S.S. (2009). From cookbook to collaborative: transforming a university biology laboratory course. American Biology Teacher, 71, 548–552.
Jain, N. & Van Staden, J. (2007). The potential of the smoke-derived compound 3-methyl-2H-furo[2,3-c]pyran-2-one as a priming agent for tomato seeds. Seed Science Research, 17, 175–181.
Lawson, A.E. (2010). How many scientific methods exist? American Biology Teacher, 72, 334–336.
Van Staden, J. (2010). Plant growth regulators from plant-derived smoke – an overview. Keynote address delivered at the Postgraduate Symposium, Department of Botany and Plant Biotechnology, University of Johannesburg, 29 October 2010.