A study on the effect of smoke water on the germination and growth o f eucalyptus pilularis

Australia, known for its frequent occurrence of bushfires during the summer season, experiences significant impact on its plant life. As a result, various indigenous Australian plant species residing in fire-dependent ecosystems have developed unique reproductive mechanisms to cope with fire-related factors. These adaptations, influencing the process of germination, can be categorized as either physical, arising from the intense heat of bushfires that triggers seed germination, or chemical, resulting from a blend of diverse chemical components present in smoke that stimulates the germination process.

The objective of this laboratory experiment in the field of biology is to investigate the impact of a solution called smoke water, comprising a combination of water, charred vegetation, and hay, on the process of seed germination and subsequent growth of Eucalyptus pilularis. Eucalyptus pilularis, commonly known as gumnut or blackbutt, is an indigenous Australian plant species that predominantly thrives in forest environments subject to regular burning events.

When compared to de-ionized water, can smoke water encourage the germination and post-germination growth of Eucalyptus pilularis seeds?

The post-germination growth of the Eucalyptus pilularis seeds by the smoke water will be more successful as a result of the smoke water's ability to germinate more Eucalyptus pilularis than de-ionized water. The height of the seedling that emerges serves as a measure of effectiveness in this experiment. from the gumnut seed that has sprouted. Eucalyptus pilularis will start to emerge from its latent state if the various substances, such as phosphorous and nitrogenous compounds contained in the smoke-filled residues of organic materials, act as chemical triggers. Since de-ionized water lacks the naturally occurring phosphorus and nitrogenous chemicals found in organic debris, such as NaN0₃, KN0₃, NH₄Cl, and NH₄N0₃, smoke water is expected to germinate.

The trees used to produce the gumnut seeds were found in nearby forestry operations. It was deemed vital to test whether or not the gumnut seeds would sprout. 

• Ten seeds each were placed in each of five potting mixture-filled Petri dishes, totaling 50 seeds.
• Each dish was given 10 ml of deionized water and kept at room temperature for two weeks.
• The number of seeds that germinated was counted at the conclusion of the two weeks.

Number of seeds germinating = \(\frac{22}{50}\)

Percentage germination = 44%

The supply of seeds was considered viable enough to proceed with the experiment.

• 10 Petri dishes
• 100g of "Yates premium quality" potting mix
• 5.00g of hay
• 5.00g of Eucalyptus leaves
• 5.00g of grass
• Electronic weighing scale (±0.01g)
• 100 seeds of E. pilularis that are 2.00 mm in diameter (±0.5mm)
• 10.0cm ruler (±0.5mm)
• 100ml of de-ionized water to create the smoke water
• 100ml of de-ionized water to create the control
• Tea strainer
• 3 × 250ml graduated beaker (±0.4mL)
• Matches
• 2 Sand baths
• 2 thermometers (±0.05°c)

• One 250 ml beaker should include 5g of hay, grass, and eucalyptus leaves.
• Light a match to ignite the organic material, causing it to catch fire. Until they are fully burnt, let them burn.
• Using the second 250 ml beaker, weigh 100 ml of deionized water. Fill the first beaker with the leaves, hay, and twigs with this water, and allow to steep for five hours.
• Using the tea strainer, pour the smoke water combination into the third measuring beaker, making sure to collect just the liquid residue.

When compared to de-ionized water, can smoke water encourage the germination and post-germination growth of Eucalyptus pilularis seeds?

• The sand baths should be heated to 30 degrees Celsius, and a thermometer should be placed in each one to confirm the temperature setting.
• Put the remaining 5 Petri dishes in one sand bath and the first 5 in the other. One will serve as our test, and one will serve as our control.
• Using the electronic weighing scale, weigh out 10 × 10.0g of the potting mix, and then put 10.0g into each of the 10 Petri dishes. 5 dishes are used to treat smoke water and 5 dishes are used to treat de-ionized water.
• Plant 10 gumnuts in each Petri dish, and then consistently bury them 0.5 cm deep in the potting soil. Place the seeds near the Petri dish's edges so that they may be seen through the glass without having to be disturbed in order to be viewed.
• For fourteen days, at 8:15 a.m., water the control sand bath with 10 ml of de-ionized or smoke water.
• Using a 10.0 cm ruler, measure the height of the emergent seedling in the test and control groups after 14 days to determine how many seeds germinated (identified by the emergence of the seedling). The height of the seedling is calculated from the soil's surface to its highest point.
• To confirm there is enough data, repeat the setup once again.

• Each dish receives the same amount (10ml) of liquid at the same time (8:15am) every day for the next 14 days.
• The 100 E. pilularis seeds used in this experiment were all kept within a size range of 2.00 mm in diameter.
• The smoke water was made from de-ionized water, just as the control group, ensuring consistency between the two groups.
• The sand baths maintained the temperature of the seeds at 30.0°C.
• The seeds were planted in the same brand of potting soil—"Yates premium potting mix"—with a consistent weight of 10.0g.
• Because the experiment was carried out in the same place on the same days, it was expected that each plant would receive the same amount of light.
• In the Petri dish, the seeds were inserted 0.5 cm deep into the soil.

The experiment lasted fourteen days in order to provide researchers enough time to evaluate the impact of the manipulated variable, the various types of water. According to the procedure, two identically heated sand baths are put next to one another, with the assumption that they are both receiving the same quantity of light. Since all samples of the potting mix came from the same batch, it was reasonable to conclude that the ingredient ratio was the same. To enable observations to be performed through the glass without disturbing the seeds, the E. pilularis was also immersed into the potting mix at a consistent depth of 0.5 cm and closer to the edges of the Petri dish.

In order to collect data for this experiment, we counted the number of seeds from various Petri plates that successfully germinated in the control and test groups, respectively. This is accomplished by checking the Petri dish's side to see if the seed coat has broken and the seedling has emerged. The height of the seedlings of the germinated seeds after the experiment's 14 days is another technique to gather data. This height is measured from the soil's surface to the seedling tip. The t-test for seedling development and the x² test for seed germination were used to compare smoke water and de-ionized water, respectively.

• Because the seeds will be arranged side by side, the light is of the same intensity.
• The contaminants present in the de-ionized water are the same.
• Each of the potting mix's component parts is present in the same quantity.
• Both sets of seeds will be exposed to the same pollutants and chemical components in the air.
• Every gumnut seed contains the same proportion of each ingredient.

• The brownish-black E. pilularis seeds had a maximum diameter of 2 mm. No clear evidence of earlier germination or shattering of the seed coat was present.
• The de-ionized water couldn't be confused with the smoke water. The de-ionized water was clear, as one could anticipate from filtration. The smoke water, on the other hand, took on a blackish straw colour as a result of absorbing the charred remains of the organic material that had burned.
• With the smoke water, definite germination was observed on a lot more seeds than with the deionized water.
• On average, E. pilularis seedlings exposed to smoke water germinated more quickly than those exposed to de-ionized water. Seven days after being soaked in smoke water, seeds began to germinate and the seed coverings began to crack, allowing the seedlings to emerge. The de-ionized moistened seeds, in contrast, took up to 10 days to begin to germinate.
• Larger seedlings usually emerge from the split seed coat of the E. pilularis that were germinated by the smoke water.
• The seed coat of the E. pilularis that was watered with the smoke water cracked noticeably more, providing greater room for the seedlings to grow and extend outside of the shell.
• The seedlings in both trials were a characteristic dark purple colour, and only the smoke water experiment's leaves developed; these were just two little, juvenile leaves, each measuring no more than about 50.0mm.

The number of seeds that clearly displayed shattering of the seed coat and the emergence of the seedling for both the smoke water and the de-ionized water test groups were counted and entered into the table below in order to calculate the number of seeds that were successfully germinated. A has a presentation of the raw data.

We can easily observe that smoke water germinates, on average, from the processed data that tells us how many seeds successfully sprouted.

An X² test was performed to see whether there was a discernible difference between the germination of seeds treated with smoke water and seeds treated with de-ionized water.

Null Hypothesis: Gumnut seeds do not become germinated when exposed to smoke water.

Alternative Hypothesis: Gumnut seed germination is impacted by smoke water.

Proportion of seed germinating = \(\frac{137}{200}\) = 68.5%

Proportion of seeds not germinating = 100 – 68.5 = 31.5%

Expected number of smoke water treated seeds to germinate = 68.5% of 100 = 68.5

Expected number of de-ionised water treated seeds to germinate = 68.5% of 100 = 68.5

Expected number of smoke water treated seeds not to germination = 31.5% of 100 = 31.5

Expected number of de-ionised water treated seeds not to germinate = 31.5% of 100 = 31.5

Number of degrees of freedom = (rows – 1) × (columns – 1) = (2-1) × (2-1) = 1

X²_calc = 3.84 for p = 0.05

We must reject the null hypothesis and accept the alternative hypothesis since the test result for X² _calc = 35.25 is significantly higher than the critical value for the null hypothesis, which is X² _crit = 3.84. If p < 0.001, the test value is significant.

Depending on the type of water it received—de-ionized or smoke water—this section of the experiment is meant to assess the gumnut seed's ability to germinate. The height of the seedling that emerged following the germination of gumnut seeds served as a gauge of effectiveness. The influence of water on germination increases with seedling height. A has the raw data.

When looking at the processed data at first glance, it is evident that smoked water has a larger average seedling height than de-ionized water and a lower standard deviation. This shown that the seedlings of the smoked water seeds outgrew those of the de-ionized water. The error bars in the graph below imply that there might be a sizable variation in how the therapy impacts seedling growth. However, the standard deviations show a wide range of variation in the outcomes, particularly for the studies on deionized water treatment. An analysis of the data using a t-test was done to confirm this.

In order to statistically test whether the shoot of smoke water germinated gumnut seedlings grew more than the de-ionized water, a two-tailed t-test for independent samples was carried out to investigate whether there is a significant difference between the growth of the seedlings.

• Null Hypothesis - The post-germination growth of the gumnut seedlings is unaffected by the smoke water.
• Alternative Hypothesis - The smoke water does affect the growth of the gumnut seedlings after germination.

t-test formula:

\(t = \frac{|\bar{X}\ -\ \bar{X_2|}}{\sqrt{\frac{S_1^2}{n_1}\ +\ \frac{S_2^2}{n_2}}}\)

degrees of freedom = n₁ + n₂ – 1 = 198

t_calc = 17.4

t_crit (p = 0.05) = 1.97

We may accept the alternative theory that the smoke water greatly increases the growth of the germinated gumnut plants because our test t value, t_calc = 17.4, is more than the crucial value, t_crit =1.97 at p = 0.05. If p < 0.001, the test value is significant.

Even though the same brand and same quantity of potting soil were used for both seeds in the experiment, the potting soil may have contained impurities that could have enhanced or decreased the ability of the seeds to germinate. This is especially likely given that the Yates brand "Contains trace elements to add extra vital nutrients". However, the seeds watered with de-ionized water might not have had this potential issue. Some of the chemicals from the smoky water also could have potentially reacted with some of the components of the potting mix and rendered them worthless. I could have used an alternative seed support, like cotton wool or filter paper, to make this better.

You would get different chemicals if you used different kinds of leaves, twigs, and hay to make the smoke water because each has a varied makeup of chemicals, some of which might be helpful for germination and some of which wouldn't. I could have used hay as the sole variable in this experiment instead of twigs and leaves. As a result, my search for answers would be more focused, and I might be able to identify the precise chemical—or the source of the chemical—that enables gumnuts to germinate successfully. For instance, it might be discovered that leaves improve seed germination whereas twigs don't. By identifying the component that best promotes seed germination, additional research might be done to determine the precise chemical that best promotes seed germination.

In addition, rather of using gumnut seeds that were all the same diameter, I could have used ones that were all the same weight. I attempted to use gumnut seeds that were only 2.00mm in diameter, but it would have been better to use seeds that were uniform in weight, such as those that weighed 0.2g, as I could have assumed that each seed contained the same quantities and makeup of nutrients, enzymes, and other chemicals.

I may have studied the effects of other smoke water concentrations as well to further focus my investigation. I could have tested a ratio of 1:5, using 1 part twigs, hay, and leaves to 5 parts de- ionised water, instead of simply utilising a 1:10 ratio of twigs, hay, and leaves to 1 part to 10 parts de- ionised water. This experiment would benefit from determining the ideal smoke water content since better and more accurate findings could be produced.

According to the results of the experiment, smoke water will effectively germinate more Eucalyptus pilularis than de-ionized water. This proved my hypothesis. Due to the substantially taller Eucalyptus pilularis seedlings that were exposed to the smoke water, it was also discovered that the smoke water was more effective at growing the Eucalyptus pilularis seeds than the de-ionized water. This may be due to the various chemicals, such as phosphorous and nitrogenous compounds, which acted as chemical triggers for the E. pilularis to begin its germination out of its dormant state and stimulate its subsequent growth in the smoke-filled remnants of the burnt organic matter (in my case, the burnt leaves, hay, and twigs). The majority of the compounds in the smoke-water mixture (NaN0₃, KN0₃, NH₄CI, and NH₄N0₃) are water soluble, making them easily absorbable by gumnut seeds. Once inside the seed, these so-called "chemical triggers" are used to initiate germination, though not all of the active substances in smoke have yet been identified. When the seed detects these varying quantities of phosphorous and nitrogenous compounds, it stimulates the germination of the seed. These chemical triggers act by changing the levels of chemicals that the seed maintains in homeostasis. Butenolides are a class of chemicals with proven germination-promoting properties. Some plants create these butenolides when exposed to high heat and smoke from wildfires. Botanists Flematti, Ghisalberti, Dixon, and Trengove in particular isolated a specific butenolide known as 3- methyl-2H-furo[2,3-c]pyran-2-one, which was discovered to cause seed germination in plants whose reproduction is fire-dependent, like the E. pilularis employed in my experiment3. Light, Berger, and van Steden provided us with one theory regarding how this butenolide, known as 3- methyl-2tf-furo[2,3-c]pyran-2-one, is formed by the plant. They proposed that this specific butenolide was created from cellulose within the plant, and that the substance, created by the cellulose, stimulated the seeds reproductive cycle and, therefore, germination4. Further evidence that my hypothesis was correct can be found in the two pie graphs that show the percentage of seeds that germinated in the smoke water experiment and the de-ionized water experiment, respectively. 88% of the smoke watered seeds germinated successfully compared to only 47% of the de-ionized water seeds, proving that my hypothesis was correct. This was supported by my 2 test, which correctly found that we could reject the null hypothesis—that the smoke water successfully germinated more seedlings than the de-ionized water—with a 95% degree of confidence. The t-test on seedling development reveals that the smoke water significantly benefits gumnut seedling growth.

Yates Gardening Ltd Sydney Australia http://www.yates.com.au/products/pots-and-potting-mix/all-purpose- potting-mix/yates-premium-potting-mix/ Last visited July 10 2011

Gavin R. Flematti, Emilio L. Ghisalberti, Kingsley W. Dixon and Robert D. TrengoveA Compound from Smoke That Promotes Seed Germination http://www.sciencemag.org/content/305/5686/977 Science 13 August 2004: Vol. 305 no. 5686 p. 977Published Online July 8 2004

Marnie E. Light, Barend V. Burger and Johannes van Staden Formation of a Seed Germination Promoter from Carbohydrates and Amino Acids http://pubs.acs.org/doi/abs/10.1021/jf050710u Agric. Food Chem., 2005, 53 (15), pp 5936–5942 Publication Date (Web): July 1, 2005