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The Effect of different Gibberellic Acid on the shoot growth of Mustard Seeds (Brassica nigra) B.B.C. Jeyashankar 7C1

Aim: The aim of this experiment is to investigate the effect of seed germination (in terms of shoot growth in mm) when different concentrations of Gibberellic acid (concentration measured in ppm) are used.

Hypothesis: I predict that as the concentration of gibberellic acid increases, the plant shoot height (mm) will also increase in terms of growth.

Variables: There are three different types of variables that were used in this experiment - The independent variable - This is the one variable that you change to find out the effect that this variable has on the experiment. - The independent variable was the different concentrations of gibberellic acid. The concentration of gibberellic acid was measured in parts per million. Use five concentrations; 20ppm (parts per million), 40ppm, 60ppm, 80ppm and 100ppm. - The dependent variable - This is the one that is contingent on the independent variable. - The dependent variable is the shoot of the height (in mm) of the mustard seed. The height of the shoot was measured from the top of the seed till the tip of the shoot - The controlled variable - These variables remain unchanged throughout the process of the experiment to ensure a fair test between sets of data along with the validity of the test showing how there were different experimental values due to the independent variable only. - The controlled variables were - The amount of duration of time of the experiment (from March 8 to March 16)

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The same type of mustard seeds (Brassica Nigra) from the same packet - The amount of solution added to each petri dish - 6cm3 was added on the first day. On the second day add 4cm3 and everyday after that add 2cm3 . - Also, place the seeds in a similar way. Every seed has its isolated location and must be placed between a cotton mesh and a paper towel. - Make sure to use the same kind of Petri Dish - Water the plants at the same time so that the duration of time that the lids of the petri dish are opened for is the same for all - Same thickness of paper towel. Folded twice. These were the controlled variables that were not decided by us but the petri dishes were all together in the same conditions to ensure that they all were at the same - Temperature (roughly 26 °C) - Humidity - Pressure - Same amount of daytime (roughly 12 hours and 30 mins)

Method: 1. Obtain six freshly cleaned Petri dishes all of the same size. 2. Label both the Petri dish and the lid as either 20ppm, 40ppm, 60ppm, 80ppm or 100ppm. There is one petri dish that is the control and you label it H  2O. 3. Place a thin sheet of cotton mesh on the bottom of the Petri dish so that the mesh is spread out all cover the bottom of the dish 4. Using a tweezer, take 10 mustard seeds (Brassica Nigra) on the cotton mesh and ensure that they are all spread out. 5. Using a syringe, put 4ml of solution of the specific solution to avoid cross-contamination ( 20ppm, 40ppm, 60ppm, 80ppm or 100ppm) and squeeze it all over the seeds. Do the same with the water petri dish (control). When taking 4ml out of a bottle, take more than 4ml in the syringe and face the tip of the syringe upwards. Flick the tip of the syringe to allow the air bubbles to come to the tip and then squeeze the syringe out so that there is 4ml left in the syringe. Spread the solution all over the petri dish 6. Once the solution has been poured all over, cover with a thin paper towel making sure all the mustard seeds are covered. 7. Repeat the same process of obtaining the same solution in the syringe but this time only 2ml is needed to cover the top

8. Once this step is complete, cover petri dishes with a lid and place them all in the same room with the same conditions, such as temperature and humidity. Use a rubber band to ensure that the petri dish is closed 9. Add in 2ml of the specific concentration of gibberellic acid (and water for the control) everyday for 4 days. As the shoot starts to grow, take off the top layer of paper towel and keep adding 2ml of gibberellic acid everyday for another four days 10. By the end of the eight day cycle, gently take off the shoots from the cotton mesh and measure the length of the shoot from the top of the seed to the tip of the shoot. 11. Use the longest 5 seeds for the processed results and calculate the mean.

Diagram of the seeds and how they should be spread out

Results: Raw Results (Raw)

Shoot Height (mm)

Conc. of G.A (ppm)

1st 2nd 3rd 4th

5th 6th 7th

0 (H2O)

49 40 40

37

36 31

/

20

63 64 47

36

34

/

/

40

68 88 72

80

74

/

/

60

87 69 70

74

75 51

80

57 67 33

55

56 46 42

100

60 88 53

64

60 68

/

/

*NOTE: The number of results received in the raw data is different for each petri dish as some seeds didn’t grow within the 8-day cycle

Processed Results (Processed)

Shoot Height (mm)

Conc. of G.A (ppm)

1st

2nd 3rd 4th 5th

Mean

S.D

0

49

40 40 37 36

40.4

±4.4

20

63

64 47 36 34

48.8

±12.79

40

68

88 70 80 72

75.6

±7.42

60

87

69 70 74 75

75.0

±6.41

80

57

67 46 55 56

56.2

±10.55

100

60

88 64 60 68

68.0

±

Conclusion: For our experiment, we had to find out how different concentrations of Gibberellic acid affected the growth of mustard seeds (Brassica Nigra). My hypothesis was that as the concentration of Gibberellic acid increased (ppm), the plant shoot growth increased as well. Through my processed results, I found that my hypothesis was incorrect. My results showed a pattern with one exception of an anomaly. I found that from 0ppm (H2 O) to 40ppm, the average shoot growth increased from an average of 40.4mm to 75.6mm  . From 40ppm  to 60ppm  , the shoot height decreased from an average of 75.6mm to 75mm and this was a similar result even though the concentration of Gibberellic acid increased. From 60ppm to 80ppm, there was a significant decrease in shoot growth from an average of 75mm to 56.2mm. And finally there was an increase in shoot growth from 80ppm to 100ppm from an average shoot height of 56.2mm to 68mm. From these results, I conclude that the Brassica nigra h ad an optimum shoot growth at 40ppm to 60ppm. The reason for this conclusion is due to the very similar average shoot height growth (75.6mm and 75.0mm respectively)with both concentrations of Gibberellic acid.

Discussion: For a seed to germinate, it needs water, oxygen and sunlight. Gibberellic Acid is a plant hormone that causes a plant to grow bigger than usual. It promotes germination, causes expansion and proliferation of the mustard seed shoot, breaks seed dormancy, and acts as a growth regulator which stimulates stem elongation. A study on Gibberellic acid that was conducted in a university in India found that the application of Gibberellic acid on plants causes an increase in the rate of photosynthesis and an increase in cell division. As a result of this, the plant will absorb more CO2, and this will cause the plant to synthesise sugars faster as the cells are dividing quicker. It can grow faster, and as a result of this, the cells will divide faster. This is because the Gibberellin is regulated in the undifferentiated cells of the plant, and this will lead to the formation of more cells. This cycle will continuously occur increasing the rate of growth of the mustard seed shoot. After completing all of the data analysis, the results from this investigation allowed me to identify which concentration of Gibberellic acid was most effective and produce the highest average shoot growth. Gibberellic Acid is a plant hormone that causes a plant to grow bigger than usual. It promotes germination, causes expansion and rapid growth (proliferation) of the mustard seed shoot, breaks seed dormancy, and acts as a growth regulator which stimulates stem elongation.

When looking at the results, it is clearly seen that at 0ppm (standard H2O) there is the least amount of average shoot growth of the mustard seeds. The average length of mustard seed shoot height was 40.4mm. This is the lowest result we got because there is no Gibberellic acid present and therefore there is nothing to break the dormancy of the seed shoots and initiate germination. However, without gibberellic there is still germination occurring but at a much slower rate as there is no hormone to speed up the rate of growth. This is the control to show how the growth of mustard seeds normally is and by using this experiment, we can analyse how Gibberellic acid changes the rate of shoot growth of mustard seeds. By using a control experiment, we can eliminate the theory that other variables were the cause for the rate of change of mustard shoot seed growth as we kept everything constant in all other experiments just like we did with our control. For our 20ppm, we got an average shoot growth of 48.8mm. This already shows that Gibberellic acid is having a positive effect of the rate of growth as it was in the same conditions as the control. The reason for the little change in the rate of growth is due to the small concentration of Gibberellic Acid present. A small concentration is more ideal than a high concentration of gibberellic acid as it helps with the growth of the shoot whereas a high concentration will inhibit the mustard seed shoot growth. The concentrations of gibberellic acid which produced the optimum growth of the shoot was at 40ppm and 60ppm. Gibberellic acid must be used in small concentrations to help regulate the growth rather than inhibit it. This  is consistent with the findings of Lovell and Booth, who also determined that gibberellic acid positively affects the shoot growth of plants (Lovell & Booth, 1967). Also through another finding, we found that their plant height was highest when they used a concentration of 50ppm (A. Akter et al). This is the optimum concentration range and it's not too low nor too high. If it was too low, the gibberellic acid given to the seeds wouldn’t have an effect and there wouldn’t be enough GA to break the dormancy of the seeds and help with the elongation of the shoot. If the concentration was too high, then the massive amount of GA given to the seeds to flood it and instead of helping with the shoot growth, it would inhibit the shoot growth and as a result, the rate of growth of the mustard seed shoot will decrease. At 80ppm, we expected a decrease in shoot height but the average shoot height was much lower than what we were expecting. Although a high concentration of Gibberellic acid will decelerate the rate of the mustard seed shoot growth and take longer to break dormancy of the seed which will cause the average shoot height of the mustard seed to decrease, we don’t expect the shoot growth to decrease so much as it

has done in our experiment. We assume that this whole petri dish containing the 80ppm of Gibberellic acid is an anomaly. The average root height at 100ppm has decreased to 68.0mm from 75.6mm in the 60ppm petri dish. This is showing us that as the concentration of the Gibberellic acid increased after 60ppm, the average rate of the mustard seed shoot growth deceased, because after this point, extra concentration of Gibberellic acid had a negative effect on the seeds causing it to build resistance to Gibberellins thus not effectively boosting plant growth.

Evaluation: While this experiment was designed to be as fair as possible with all the variables kept constant other than the independent variable, there were some limitations in the experiment. Keeping all the controlled variables the same ensured that the experiment was as fair as we could make it and this could be repeated and give very similar results. Keeping the controlled factors constant throughout the experiment was important and any changes to these variables could favour plant growth to one plant and not the other. Our experiments were all conducted from the 8th March to 16th March. We had to restart twice. The first restart was due to us mixing different concentrations of Auxin. The second time was due to the lockdown. We initially used auxin to compare the mustard seed shoot growth but due to lockdown, we could not add more Auxin solution and therefore our experiment was not considered as viable. We then decided to use Gibberellic Acid to ensure that we could add in solution everyday and make sure that the rate of growth of the mustard seeds shoots were changing. By restarting, lockdown did not have an affect on the results we obtained. When we were measuring the shoot height of the mustard seed, we had to prevent zero error when using the ruler. When taking the readings we used a ruler that had a marking at 0mm and so we prevented zero error when taking measurements. By doing this we are able to keep consistent methods of measuring the shoots. If we did not do this, then we would measure some shoots longer than what they are and some shoots shorter than what they are and this would not give us accurate results. We ensured that we added the same amount of solution to every petri dish. We did this by using the same procedure of obtaining the solution in the syringe. For example, on the first day we added 6ml of the specific Gibberellic acid concentration to every petri dish. To obtain this 6ml, we used a syringe and drew out 8ml of the specific

Gibberellic acid solution. We then faced the tip of the syringe upwards and flicked it using a finger. This made sure that all the air bubbles that were in the syringe, moved towards the tip of the syringe. Then keeping the syringe facing upwards, we pushed the plunger so that there was only 6ml of solution left in the syringe. This constant procedure was carried out whenever we added solution and this ensured that a fair test was being carried out. Also, when we were looking at the syringe, it was directly at eye level so that we could prevent parallax error. If we did not look at it directly at our eye level, we would’ve added too little or too much solution and it would not have been a fair test. If this wasn’t consistent, there is a possibility that the seeds wouldn’t have received the same amount of Gibberellins compared to other seeds, meaning that the average height of the shoots would have been decreased. The petri dishes were all together in the same class room and next to each other to ensure that they all experienced the same temperature, humidity levels, pressure and amount of daytime. An ideal way to have kept the temperature, humidity levels, pressure and sunlight constant, a controlled greenhouse should’ve been used. This meant that all petri dishes were at, for example 26 °C, 70% humidity levels, received 11hrs and 30mins of sunlight, and also was at standard atmospheric pressure (1atm). In our experiment our petri dishes were stacked one on top of eachother. This meant that some petri dishes received less sunlight than others and so that was a possible factor which contributed to our anomalies. Another ideal situation would be to conduct this whole experiment again about a week or two later. This will ensure that the results that we obtained in each petri dish was similar and that firstly, the experiment was repeatable and secondly that we obtained fair results and no anomalies were taken. We used the seeds from the same jar containing all the mustard seeds. However we don’t know if all these mustard seeds are from the same packet or not. This could easily differentiate the results of the average shoot growth slightly and therefore could’ve been a limiting factor for our experiment.

Comparison:

The results above were obtained by Albhaisi and his group. As you can see the results that we got compared to Albhaisi’s group are very similar. Both of ours showed a peak in the 40ppm-60ppm region and a bell curve was formed. We can also conclude that our 80ppm experiment was an anomaly as there is a gradual decreasing trend in the average shoot height of the plants. However our 80ppm petri dish gave a significantly low average value messing up with the trend.

Our experiment also shows a very similar trend with an external source. This is an experiment conducted by Int. J. Sustain and his results showed a bell curve in terms of

average height of the plant. Although this isn't directly related to ours because we did the shoot height, it still is relatively similar when compared to our findings. Overall, this experiment was conducted in such a way that it can be repeated and similar results can be achieved. We’ve received results that can be backed up by other sources from different studies on the Brassica nigra  (mustard seed). Although our hypothesis was incorrect, we have identified the relationship between the average mustard seed shoot length with different concentrations of Gibberellic Acid.

References: 1

"Plant growth studies mineral nutrition growth regulators and the effect of ...." Plant growth studies mineral nutrition growth regulators and effect of abiotic stresses. Accessed 17 Mar. 2021. 2 "Gibberellic Acid for Fruit Set and Seed Germination - Power Grown." Gibberellic  Acid for Fruit Set and Seed Germination. Accessed 18 Mar. 2021. 3 ”The Effect of Gibberellins on Growth and Flowering of Fragaria and Duchesnea C.G Guttridge and P.A Thompson”. https://www.jstor.org/stable/23686408?seq=1  4 "Effect  of auxin and gibberellic acid on growth and yield components ...." Effect of auxin and gibberellic acid on growth and yield components of linseed (Linum usitatissimum L.). Accessed 17 Mar. 2021 Zhu, G., An, L., Jiao, X., Chen, X., Zhou, G., & McLaughlin, N. (n.d.). Effects of gibberellic acid on water uptake and germination of sweet sorghum seeds under salinity stress. Retrieved from https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0718-58392019000300415

Weinstein, R. (2019, October 10). Maximizing Gibberellic Acid's Efficiency to Increase Growth. Retrieved from https://www.sciencebuzz.com/maximizing-gibberellic-acid-efficiency-to-increase-growth/

Figure 2f from: Irimia R, Gottschling M (2016) Taxonomic revision of Rochefortia Sw. (Ehretiaceae, Boraginales). Biodiversity Data Journal 4: E7720. https://doi.org/10.3897/BDJ.4.e7720. (n.d.). doi:10.3897/bdj.4.e7720.figure2f

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