A study on the effect of smoke water on the germination & growth of 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.