Influence of Light & Temperature on Lactuca sativa Seed Germination By: Craig Edwin Meredith

1. Influence of Light & Temperature on Lactuca sativa Seed Germination By: Craig Edwin Meredith Procedure Seeds of Lactuca sativa L. were purchased from Burpee, Ferry Morse Seed Company, Ed Hume Seeds, and Livingston Seed and were stored at room temperature in darkness until used. Working in limited light, petri dishes were prepared in a Forma Scientific Biological Safety Cabinet. Each petri plate contained two layers of Fisher Scientific Whatman filter paper that were placed in the lid of the petri plate. Five milliliters ultra-pure water was added to the filter paper. The variety of seeds tested was placed onto a round sheet of filter paper. Forceps were dipped into EtOH and passed through a flame. Once cooled, the forceps were used to pick up the seeds individually and they were placed on top of the soaked filter paper. Seeds were selected against deformities and discoloration, and for uniformity in size. Seeds were arranged into even columns and rows. The petri plates were then sealed around the edges with masking tape. For each variety tested in dark, there was one petri dish wrapped in aluminum foil. The petri dishes of the seeds being tested were placed into either a Ziploc Double Zipper Freezer bag (Phase I) or Seal a Meal bag (Phase II). For each test approximately six to eight dishes could fit inside either plastic bag. Petri dishes not wrapped in aluminum foil were placed on top of the petri dishes wrapped in aluminum foil and the seeds were exposed to two General Electric Ecolux Technology plant and aquarium bulbs (40 watts per bulb). The plastic bag was kept submerged in a Fisher Scientific ISOTEMP 210 water bath using a plastic grid that was tied to a metal base and a Seal a Meal plastic bag that contained pebbles and water. The light source was hung four inches above the water bath. Temperature was programmed on the water bath. For temperatures below 24°C, the experimental apparatus was set-up inside a micro-fridge. Germination was checked 24 hours later and the procedures were repeated using the same petri dishes cleaned, new seeds, and a different temperature. Figures 3 and 4, shows the germination of various lettuce types in light and darkness at 18 to 32°C in a Seal a Meal bag. In darkness and light, maximal germination was seen in almost all varieties to stay consistent between 22 to 28°C (Fig. 3, 4). At extreme conditions a decrease in germination percentage was seen in many of the tested lettuce varieties (Fig. 3, 4). Black-Seeded Simpson, Great Lakes, and Parris Island in darkness and light showed similar curves. Maximal germination within the temperature range in darkness was not always the same for all varieties. Burpee Bibb (light), Iceburg (light), and Buttercrunch (darkness and light) showed favorable amounts of germination at temperatures above 30°C. Introduction It has long been known that there are several factors that may promote seed germination. According to Negm et al. (1972, 869), ethylene and carbon dioxide have been reported to stimulate the germination of certain seeds. The growth and development of field crops are largely controlled by the effects of temperature and light (Tei et al., 1996, 645). Other known factors that may promote seed germination are oxygen, desiccation, hydration, fire, and smoke. Since the work by Borthwick et al. it is known that light is involved in the promotion of germination of many seeds (Valio et al., 1972, 677). According to Valio et al. (1972, 677), the regions of maximum effectiveness on seed germination is located at about 660 nm (red) for promotion and about 730 nm (far-red) for inhibition. Red and far-red effects are mutually antagonistic. Red and far-red effects can be repeatedly reversed by opposite radiation and the final radiation determines the response of the plant (Raven et al., 2005, 631). This light is detected by the plant by phytochrome, a cytoplasmic pigment found in green plants that absorbs light and regulates dormancy, seed germination, and flowering. Phytochrome exists in two forms, Pr and Pfr (Raven et al., 2005, 633). In unirradiated, dormant seeds, Pr (inactive form) absorbs red light or sunlight and it gets converted into an active, dormancy-breaking form known as Pfr (Bewley et Black, 1994, 238). Pfr is reverted back to Pr by dark reversion or far-red light or it can be lost through destruction that occurs over a period of time and involves hydrolysis by a protease (Raven et al., 2005, 633). According to Park Seed Co., Lactuca sativa, or lettuce, needs light to germinate the seeds. Bewley and Black (1994, 237) found that Grand Rapids lettuce seeds remain dormant in darkness only above about 23°C and germinate without illumination below 23°C. Therefore, light requirement depends on temperature (Saini et al., 1989, 311). Saini et al. (1989, 311) saw approximately one-hundred percent germination in Grand Rapids lettuce seeds held in dark conditions at temperatures up to 20°C. Above 20°C germination percentages declined with an increase in temperature reaching zero at approximately 25 to 35°C (Saini et al., 1989, 311). This research is an attempt to understand the germination behaviors of other varieties of lettuce seeds when exposed to a range of temperatures and light or darkness. Figure 1: Germination of Grand Rapids at various temps. in darkness and light (n=100). (Saini et al., 1989, 313) Figure 2: Germination of Grand Rapids Exposed to Darkness and Light (n=20 and n=50) • Varieties • Black-seeded Simpson Grand Rapids • Burpee Bibb Iceburg • Buttercrunch Parris Island • Great Lakes Figure 3: Germination of Lettuce Seeds in Light and Darkness (n=50) Figure 4: Germination of Lettuce Seeds in Light and Darkness (n=50) Discussion Not all varieties of lettuce tested responded the same to darkness, light, and temperature. The hypothesis cannot be supported based on the data. All varieties of lettuce seed in darkness below 23°C did not show maximal germination and as temperatures increased germination not always decreased in darkness. Therefore, the data is not a direct representation of what the tertiary sources suggest. Although the data for Grand Rapids lettuce seed was not exactly the same as compared to that of Saini et al. experiment, the experimental apparatus configured did produce some similar characteristics. Looking at the germination characteristics of different varieties and their response to darkness and light, Burpee and Iceburg showed considerable differences in darkness and light at higher temperatures. Errors in the experiment that could have resulted in some outlying points are during preparation of petri dishes enough red light could have been absorbed by the seeds before wrapped up in aluminum foil, therefore converting the Pr into the active Pfr and producing a biological response. According to Saini et al., 1989, 311), flashes of light can stimulate a dormant seed, only to certain temperatures. In this experiment most lettuce seeds showed a biological response between 22 to 28°C. Negm et al. (1973, 1089), reported that Great Lakes has been considered light-insensitive because they germinate equally well in light or darkness at 20 to 25°C and at 35°C under either condition germination is inhibited. Further research needs to be done on the response of other various lettuce varieties to darkness, light, and temperature. Very little information on other varieties was found. Another factor that could have affected the results was in Phase I the Ziploc Double Freezer bags were used and for Phase II the Seal a Meal bags were used. In Figure 2, there was a clear difference between darkness and light using Ziploc Double Freezer bags, whereas using the Seal a Meal bags (Fig. 2, 3, 4) smooth curves were not seen. The Seal a Meal bags did work better to keep out water, but this could have affected the results. It might be possible that the plastic bag could have emitted ethylene; which ethylene is known to increase seed germination (Abeles et Lonski, 1969, 277). Phytochrome controls ethylene production. According to Abeles et Lonski (1969, 277), seeds treated with red light produce more ethylene. This could be the reason why maximal germination was seen above 23°C in the experiments using the Seal a Meal bags. There could also be a difference in the amount of light that passes through the Seal a Meal and Ziploc Double Freezer bags. Future Experimentation Future experimentation using this experimental apparatus, one should see if the data is consistent through multiple trials. If someone were to repeat this experiment, they should increase the sample size to 100 and test to see if there is a difference in data using different plastic bags. It would also be nice to find other varieties of lettuce seed and compare suppliers of a certain type by doing a t-test or ANOVA. Future literature on this subject could help those who have an interest in cultivating lettuce to produce large product yields. Results Figure 1 shows the germination for data of Saini et al. Grand Rapids at various temperatures in darkness. The legend for Figure 1 indicates the germination of lettuce seeds in the dark and in response to red light treatments given as a single irradiation (SI) or repeated irradiations (RI) at different frequencies. Above 27°C in the dark few Grand Rapids lettuce seed germinated (Fig. 1). In Figure 2, the data collected from both phases on Grand Rapid lettuce seed germination were superimposed. Maximal germination percentage of Grand Rapids exposed to darkness was not seen until 24°C in both plastic bags used (Fig. 2). Figure 2 shows that at cool temperatures, seeds exposed to darkness can decrease germination and at higher temperatures similar results can occur. Grand Rapids in the Ziploc Double Freezer plastic bags showed a sharp decrease in germination percentage as temperatures increased above 24°C in darkness and at 25.5°C in light (Fig. 2). Grand Rapids in the Seal a Meal plastic bag did not show a gradual decrease in germination percentage as temperatures increased above 24°C in darkness and light (Fig. 2); instead a drastic decline in germination percentage occurred between 30 to 32°C (Fig. 2). • Experimental Strategy • Phase I Test germination of Grand Rapids lettuce seeds • n = 20 seeds • Range of temperatures (24°C - 35°C) • Light versus Darkness • Ziploc Double Zipper Freezer plastic bag • Phase II Test germination in other lettuce varieties • n = 50 seeds • Range of temperatures (18°C - 32°C) • Light versus Darkness • Seal a Meal plastic bag References & Acknowledgements 1. Abeles FB, Lonski J. 1969. Stimulation of lettuce seed germination by ethylene. Plant Physiol. 44: 277-280. 2. Bewley JD, Black M. 1994. SEEDS Physiology of Development and Germination Second Edition. New York and London; Plenum Press, 237p. 3. Negm FB, Smith OE, Kumamoto J. 1973. The role of phytochrome in an interaction with ethylene and carbon dioxide in overcoming lettuce seed thermodormancy. Plant Physiol. 51: 1089-1094. 4. Park Seed Co. 2007. Geo. W. Park Seed Co., Inc. 5. Raven PH, Evert RE, Eichhorn SE. 2005. Biology of Plants, 7th Edition. W.H. Freeman and Company Publishers. 6. Saini HS, Consolacion Ed, Bassi PK, Spencer MS. 1989. Control processes in the induction and relief of thermoinhibition of lettuce seed germination actions of phytochrome and endogenous ethylene. Plant Physiology 90(1): 311-315. 7. Valio IFM, Kirszenzaft SL, Rocha RF. 1972. Germination of achenes of Bidens pilosa L. I. Effect of light of different wavelengths. New Phytol. 71: 677-682. I would like to thank Dr. Almuth Tschunko, Biology Department, research advisor, who provided support and insight for this project. I would also like to thank the rest of the Biology Department Faculty and the Senior Capstone Class ’07. • Hypothesis • All varieties of lettuce seed in darkness below 23°C will show maximal germination; as temperatures increase above 23°C germination decreases in the dark. • All lettuce varieties will germinate when exposed in light. Marietta College