Authors: Hamza Ahmed and Anish Kalyan
Peer Reviewer: Rajit Tumala
Professional Reviewer: Levante Pap
The objective of this experiment is to observe how Multiwalled Carbon Nanotubes (MWCNTs) and the cytokinin 6-Benzylaminopurine (6-BAP) affect the growth rate of Brassica oleracea var. italica (Red Cabbage) microgreens. MWCNTs and the 6-BAP have been shown to individually increase plant growth. Literature has also shown that B. oleracea is most compatible with 6-BAP and contains the anti-carcinogenic anthocyanin which produces its purple-red hue. This year research has focused on identifying how MWCNTs in conjunction with 6-BAP affects dry weight, fresh weight, change in mass over time, anthocyanin content, and Vitamin C content in B. oleracea microgreens. In order to properly simulate standard growth conditions for microgreens, they were grown in a 12/12 light cycle using growth lights. Five B. oleracea seeds were sterilized and placed in one of the four agar compositions (repeated for each agar variation). The four compositions were as follows: 3 controls (MS Media, 6-BAP + MS Media, MWNCT+ MS Media) and 1 experimental (MWCNT+ 6-BAP+ MS Media). The microgreens were then grown for 8 days, changes in mass being collected every 2 days, and extracted to measure fresh weight. The plants were then lyophilized, and the dry weight was measured. Anthocyanin was extracted from each microgreen using a standard buffer and content was quantified through absorbance analysis using a microplate reader. Results show that the change in mass over time of the experimental group was the second most and third most in the two trials with conclusive data. The fresh weight, dry weight, anthocyanin concentration, and Vitamin C concentration data were inconclusive. Though the experimental media content seemed to have some effect on the dependent variables, its impacts were not substantial. Future experiments would involve adjusting the concentrations of 6-BAP to find the optimal concentration in the experimental agar and conducting more trials to establish the correlation between MWCNT, 6-BAP and the growth mass.
Carbon nanotubes (CNTs) are a relatively new nanomaterial in the scientific industry. They are a carbon allotrope, meaning physical form of carbon, that is in the shape of a tube. Previous research has shown how CNTs can be used in inorganic materials and what properties they possess, such as electric conductivity, thermal conductivity, and tensile strength. In the biomedical field, they have been shown to be useful in drug and gene delivery. Research is currently being conducted on their potential uses in tissue engineering and biosensing. These properties have been shown to be present in animal testing as well, but little research has been conducted on the effect of Multiwalled Carbon nanotubes on plant development and growth.
An effect that CNTs’ have in vivo is that they can increase cell growth within the organism. This has been shown in both mammals and plants, but they work in different ways. In previously conducted research, a tobacco callus was grown in Murashige and Skoog Medium containing multiwalled carbon nanotubes1. A range of concentrations of MWCNTs (multiwalled carbon nanotubes) were used from 5 g/mL to 500 g/mL. MWCNTs are similar to normal CNTs, but an important distinction is the multiple layers of graphene or carbon.The amount of growth over a period of time was compared to a normally induced tobacco callus. The results showed that the tobacco callus with MWCNTs did have 55%-64% greater cell growth than the control callus. Raman analysis showed that the MWCNTs upregulated the aquaporin gene within the cells, creating more aquaporin protein, which allowed for greater amounts of water to enter the callus over the control.
A similar experiment was carried out with external ionic nutrients and maize plants. The results were similar to the findings with the tobacco plant. The dry and fresh weight of the plant were shown to increase in the presence of MWCNT’s with the concentration of 20 mg/L.The reason that the plant growth rate was increased was due to the ionic nutrients using the increased water flow caused by the change in the aquaporin gene to get to different parts of the maize plant. A disadvantage however was that the MWCNT-ion interaction between the MWCNTs’ surface and nutrients present in the growth media introduced a redox reaction causing a change in the ions of the nutrients2.Nutrients such as Fe2+ [use superscripts and the other ions as well] oxidized into Fe3+ and nutrients such as Ca2+ became more prominent. As MWCNT concentration decreased, the increased growth caused by the ionic nutrients increased. Concentrations of 5 mg/L -60mg/L were used and the ionic concentrations showed a decreasing trend. This, however, was not the case for all ions. The interaction between the ions and MWCNTs was something that was not expected and changed the nutrients absorbed by the maize. This study also showed through SEM (scanning electron microscope) analysis that increased water delivery caused by the MWCNTs was concentrated near the area in contact with the media. The introduction of MWCNTs into the system increased the capillary action between the roots and the media allowing for greater amounts of water to be absorbed.
The majority of the experiments conducted use multiwalled carbon nanotubes over single walled or normal activated carbon. The reason for this is the difference in surface tension between the different nanoparticles. In cell biology, the smallest of changes can affect the interaction between the material, different cells, and proteins. One of the main differences between SWCNTs (single walled carbon nanotubes) and MWCNTs is their surface tension. This is due to the greater number of layers found in MWCNTs, making them more rigid and having a higher surface tension. The surface tension of the material directly affects the interaction between the nanotubes and different proteins. Tau proteins were used by Zeinabad, Zarrabian, Saboury, Alizadeh, and Falahati (2016)3. SWCNTs were shown to induce greater change in the proteins than the MWCNTs did, meaning that they affected the system more than MWCNTs. When creating a controlled experiment, the least amount of change is necessary so that other variables can be tested. This is a reason that MWCNTs are used in plant cell-based experiments over SWCNTs.
It was determined that the model organism for our experimentation would be Brassica oleracea. B. oleracea var. Italica, also known as the red cabbage microgreen, is globally consumed as a food and is commonly available in local markets, is affordable, and represents a significant source of phytonutrients in the human diet. Cabbage microgreens also have health-promoting phytochemicals and are anticarcinogenic, making them an important nutritional addition to the human diet. In addition, they have antioxidant, anti-inflammatory, and cardioprotective benefits4. In addition, the compatible concentrations of the MWCNTs found were 10, 20, 40, and 60 mg/L. Variation from these concentration, specifically concentrations above 500 mg/L, could result in toxicity to the plant. Due to its nutritive and anti-carcinogenic properties and its compatibility with the listed MWCNT concentration, B. oleracea is a suitable organism for our experimentation.
In the agricultural field, external cytokinins and hormones are used on plants in order to increase plant growth. If CNTs are to be used in the agricultural field, the effect of cytokinins on the CNT enhanced growth rate on the plant must be compared. No previous research has been conducted on the effect of external cytokinin on the enhanced growth rate caused by CNTs in plants. There has been research on the natural cytokinin amount with in the plant and how CNTs affect it5. Rice seedlings were used and different types of CNTs were added to the growth medium: MWCNTs, Fe-filled CNTs, and Fe-Co filled CNTs. The results show that internal cytokinin amount in fact decreased in response to all of the different CNTs that were tested. Cytokinins are a type of hormone that promote tissue growth and flower budding allowing for faster growth. If the internal value of cytokine decreases but plant growth rate increases with the presence of CNTs, then increasing the cytokinin concentration in the plant should increase the growth rate further.
A study has been conducted showing the effect of 6- benzylaminopurine(6-BAP) on B. oleracea. 6-BAP is a synthetic cytokinin that can increase plant growth rate in a variety of plants. This relationship has been demonstrated specifically in B. oleracea with the use of 6-BAP6. The cytokinin was shown to reduce the presence of chlorophyll degrading genes. This resulted in the plant performing photosynthesis without the reduction of chlorophyll. 6-BAP has also shown to delay chloroplast dismantling and leaf yellowing in broccoli florets. A separate article gives concentrations of 6-BAP that should be used with B.oleracea 7. The plant was grown in a solution of 6-Benzylaminopurine with the concentration of 2.45, 4.90 ,7.35, 9.80, 12.25, and 14.70 M. The results showed that the best results were found of broccoli life with the concentration of 12.25 M with 56.67% greater root growth. The higher concentrations slowed growth down. A major issue that comes about when discussing cytokinin is the denaturing of the proteins during the autoclaving process. Studies have shown8 that this does not occur in adenine based cytokinins. 6-BAP is indeed adenine-based and was used in the experiment. After an autoclaving cycle, High Performance Liquid Chromatography (HPLC) was used to test the peaks and no difference was shown. This removes the issue of denaturing of the cytokinin.
Anthocyanin is an anti-carcinogenic antioxidant, found in B.oleracea microgreens, that gives the red cabbage its red-purple hue 9. As it is unique and abundant in red cabbage microgreens, anthocyanin and its concentration varies in red cabbage based on “growth rate”. As a red cabbage microgreen grows, more anthocyanin will be present to give it the color and unique antioxidant, anticarcinogenic properties proportional to its size. For the extraction of anthocyanin from the B. oleracea microgreen, first, a 100 mg portion of lyophilized powder of the red cabbage microgreen was transferred to 2 mL of water/formic acid in a 2 mL eppendorf tube. Then the solution was vortexed for 5 minutes, ultrasonicated for 20 minutes, and centrifuged at 8000 rpm for 15 minutes. This extract was then filtered through a 0.45 m PTFE syringe filter. Detection was made at 530 nm wavelength and the quantification of anthocyanin was based on peak areas and calculated as equivalents of representative standard compounds10.
Since CNTs will be used in the agricultural environment in the near future, it is important to see their effect on the crops that will be planted and how they will be transported throughout the plant. The problem arises of how CNT’s will affect the utilization of crops and what steps can be taken to simplify the process. It has been shown CNTs tend to congregate in the roots of plants, which means that the fruit and leaves are generally untouched by the CNTs11.
Carbon nanotubes are an integral part of agriculture and they are beginning to be widely used. Many different factors, including the concentration of CNTs and CNT size, pertain to this research. Significant amount of research has been conducted on the amount of CNTs used to prevent toxicity, but many external variables have not been tested on the plant system. Therefore, the purpose of our research is to show the effects of the addition of the external cytokinin 6-Benzylaminopurine and MWCNTs on the growth rate of B.olceara. This is different from the previous research because it tests a combination of variables that has not been tested before on an organism with many commercial applications.
It is hypothesized that when both cytokinin 6-Benzylaminopurine and multiwalled carbon nanotubes (MWCNTs) are used in conjunction, these two factors will increase plant growth, and nutritional content, at an elevated rate compared to using them separately. This is justified through previous research which demonstrated that both cytokinin 6-Benzylaminopurine and multiwalled carbon nanotubes (MWCNTs) have been shown to increase plant growth and flowering rate individually. Additionally, increased plant growth is inherently tied to increased nutritional content in plants, resulting in the inclusion of this variable in the hypothesis. This hypothesis was tested through manipulations made to the media compositions that B.oleracea microgreens were grown on for 8-10 days. The independent variable was the media content which was dependent on the presence of 6-BAP (12.25 M) or MWCNTs (80 mg/L). The dependent variables were the growth rate (measured through fresh weight, dry weight, and change in mass over time), anthocyanin concentration, and Vitamin C concentration. Both anthocyanin and Vitamin C are antioxidants particularly expressed in these plants, making them good indicators of the plants’ nutritional contents. Three control media compositions were used (MS media, MS media + 6-BAP, MS Media + MWCNT) to ensure that the growth media itself and both of the independent variables (6-BAP and MWCNT concentrations in the media) had similar effects to those observed in previous literature. One experimental media composition, which is the main focus of this research, was used (MS media + 6-BAP + MWCNT) to observe the effects of the combination of the independent variables. 5 microgreens were grown on each of the four media compositions for 8-10 days, and the dependent variables were respectively collected.
Materials and Methods
In order to test whether the MWCNTs and cytokinin would affect plant growth, an experiment was designed using agar media in order to grow the red cabbage microgreens. The first step was to prepare the MWCNTs solution. Since they can be considered carcinogens, the stock solution was prepared by a certified chemist with a concentration of 150 mg/L. After the stock solution of MWCNTs was prepared, each individual erlenmeyer flask was made. The first solution was prepared by adding 4.24 g of MS media powder to 200 mL of distilled water, this gave a final concentration ½ MS media as stated by previous literature.
The cytokinin solution was then prepared by adding 2.27 mg of 6-BAP to 5 mL of Sodium Hydroxide. This was then added to 195 mL of distilled water along with 4.24 g of MS media powder. After the other solutions were prepared, the MWCNT solution was then ultrasonicated in a bath sonicator for six minutes. 80 mL of solution was poured out into an erlenmeyer flask and the remainder was filled up to 200 mL with distilled water and 4.24 g of MS media. This process was also done for the experimental group, but 5 mL of sodium hydroxide and 6-BAP solution was added with the same concentrations.
After all the solutions were made, 0.8 g of agar powder was added to all the solutions to make a 0.4% agar solution. The pH of each solution was then adjusted using hydrochloric acid and sodium hydroxide. The pH was adjusted using a pH probe and the desired range was from 5.7-5.9. The solutions and growing jars were then put in autoclave bottles and autoclaved at 121 degrees Celsius, 15 psi, and 15 minutes. Then the solutions were removed, and 70 mL of each solution was poured into a jar. A control jar was set up with any remaining solution. The jars were then lidded and cooled at 4 degrees Celsius.
After the jars cooled, the initial mass of each jar was recorded using a precise scale. About 40 microgreen seeds were then sterilized for 15 minutes in a sodium hypochlorite solution. They were then rinsed and placed in water to remove the sodium hypochlorite. Five seeds were then placed in each jar using tweezers. The tweezers were bleached after each seed in order to prevent contamination of the agar. The seeds were placed half in the agar to allow for a root system to grow, but also allow the plant to sprout upward. Each jar was the saran wrapped and taped in order to prevent evaporation of the agar. Three holes were poked into the saran wrap using tweezers to allow gas exchange.
The plants were left to grow for eight days with a 12/12 light cycle. Growth lights rated 15 watts were utilized and evenly spread out to allow light distribution to all plants. The mass of the jars were recorded every 2-3 days in order to get change in mass over time. After the growing time was completed, the plants had to be extracted. This was done by heating up the jars using a hot plate to 70 degrees Celsius for about 10 minutes. Each plant was then removed using tweezers, careful to keep the root system intact. After the plant was removed, it was placed in a label area and prepared for fresh weight measurement.
In order to measure the fresh weight, each plant was lightly rinsed with distilled water and then patted dry. If the plant was not completely dry, error would be shown on the scale due to evaporation. After all the masses were recorded, The plants were placed in labeled petri dishes and placed in a lyophilizer to prepare the samples for dry weight measurement. The lyophilizer was set for a 48 hour cycle and reached a maximum temperature of -40 degrees Celsius. After this time, the plants were removed from the lyophilizer and the dry weights of each plant was measured.
The next step was to extract and find the concentration of the anthocyanin per plant. The first step was to make the anthocyanin extraction buffer as shown in previous literature. 100 mL of solution was prepared with methanol, water, and concentrated hydrochloric acid (80:20:1). One mL was then pipetted into a set of eppendorf tubes labeled for each plant. Then, each plant was ground up with 0.5 mL of extraction buffer and added to the respective eppendorf tube. Each tube was then filled to 2mL of solution with the extraction buffer. This was then centrifuged at 8,000 rpm for 15 min. After centrifuging, each solution was filtered through a 0.45 micrometer filter syringe. Each solution was decanted, as the supernatant was required. The filtered solutions were then placed in another set of eppendorf tubes.
The next step was to measure the absorbance of each of the trials. 300 microliters of each solution was pipetted into a well plate by row. A protocol was set up on the microplate reader, measuring the absorbance at 530 nm and 657 nm. The data was collected and analysis was performed to find the concentration.
The second value that was measured was Vitamin C or ascorbic acid concentration. This value was found using an iodine titration. In order to perform the titration, the samples had to be prepared first. Since not enough mass was present to perform an individual titration per plant, each of the five plants in a trial were put together and ground using a mortar and pestle. Several sets of 10 mL of distilled water were added to make the extract a liquid solution. This was then filtered using a normal coffee filter and the volume of the solution was made 100 mL by adding distilled water.
20 mL of sample solution was added to 150 mL of distilled water in a flask along with 1 mL of starch indicator solution. A burette was filled with 0.005 mol/L of iodine solution. A titration was performed with the end point being signified by the first permanent trace of blue or black in the solution. This was then repeated with the same sample until concordant results were obtained. This was repeated for each of the four agar variations in the experiment.
After the titration was done, several calculations had to be performed dependent on the titration itself. The average volume used for the color shift in each media had to be calculated along with the amount of moles of iodine used. This could be found by knowing the molar mass and the volume of iodine used. The number of moles of ascorbic acid was then determined using the equation :
ascorbic acid + I2 -> I– + dehydroascorbic acid
Finally, the concentration was then calculated in g/100 mL for the entire microgreen solution.
In order to test whether the data collected had any significance, a non parametric Kruskal Wallis test was performed on the data. Since a low sample size was used, and not all trials had the same number of samples due to variability in the seed growth. The p- value would be used to determine whether the data was significant or not.
After the trials were completed, the materials had to be disposed off. All of the jars were autoclaved at the same aforementioned settings. The MWCNT trials were disposed of in a separate bottle to be chemically incinerated later. The remainder of the trials were placed in a bio bag and thrown away in the proper container.
Table 1: Fresh weight in grams (g) (Trials 1 and 2).
Table 2: Fresh weight in grams (g) (Trials 3 and 4).
Table 3: Fresh weight in grams (g) (Trials 5 and 6).
Table 4: Dry weight in grams (g) (Trials 1 and 2).
Table 5: Dry weight in grams (g) (Trials 3 and 4).
Table 6: Dry weight in grams (g) (Trials 5 and 6).
Mass over time
Table 7: Mass over time in grams (g) (Trial 1 and 2).
|Trial||Jar w/ Agar ONLY (g)||SEED + Jar (DAY 0)||SEED + Jar (DAY 2)||SEED + Jar (DAY 4)||SEED + Jar (DAY 6)||SEED + Jar (DAY 8)||SEED + Jar (DAY 10)|
Table 8: Mass over time in grams (g) (Scrapped).
|Trial||Jar w/ Agar ONLY (g)||SEED + Jar (DAY 0)||SEED + Jar (DAY 2)|
|Control MS jar||205.5454||205.5454||205.327|
Table 9: Mass over time in grams (g) (Trials 3 and 4).
|Trial||Jar w/ Agar ONLY (g)||SEED + Jar (DAY 0)||SEED + Jar (DAY 2)||SEED + Jar (DAY 4)||SEED + Jar (DAY 6)||SEED + Jar (DAY 8)|
|Control MS Jar||207.1586||206.8126||206.7683||206.7168||206.6743|
Table 10: Mass over time in grams (g) (Trials 5 and 6).
|Trial||Jar w/ Agar ONLY (g)||SEED + Jar (DAY 0)||SEED + Jar (DAY 2)||SEED + Jar (DAY 6)||SEED + Jar (DAY 8)|
|Control MS jar||205.4319||205.0776||204.1266||203.6764|
Table 11: Anthocyanin absorbance values at 530 nm and 657 nm (Trial 3).
Table 12: Anthocyanin absorbance values at 530 nm and 657 nm (Trial 5).
Vitamin C titration
Table 13: Iodine solution used in Vitamin C titration in milliliters (mL) (Trial 6)
Graph 1: Change in mass over time (Trial 1).
Graph 2: Change in mass over time (Trial 2).
Table 14: Anthocyanin concentration in [A]/g (Trial 3).
Table 15: Vitamin C concentration in g/100 mL (Trial 6)
Table 16: P-values showing the significance in difference between control and experimental group dependent variables.
|P-Values showing the significance of collected data|
Table 16: P-values showing the significance in difference between control and experimental group dependent variables.
A non- parametric Kruskal Wallis test was performed. The established significance value was 0.05. The p values are shown above and the significant values are due to error in control group’s dependent variable measurement, leading to faulty differences between the control and experimental groups.
Anthocyanin concentration = (A530 – (1/3)A670)/ mdry
A530= 0.537, A670=0.341, mdry=0.05088
[A]/g= (0.537- (1/3)(0.341))/ 0.05088 g= 8.3202 [A]/g
Vitamin C (ascorbic acid) concentration calculations
Difference= Value 1- Value 2
Value 1=2, Value 2=1
Difference= 2-1= 1
Average= Sum of values/number of values
Sum of values= 6, number of values=2
Average= 6/2= 3
mL to L: 1000 mL= 1 L, 1 mL= 0.001 L
Moles of I2= 0.005 mol/L x Average difference (L)
Average difference= 0.0098 L
Moles of I2= 0.005 mol/L x 0.0098 L= 4.9 x 10^-6 mol of Iodine
Vitamin C concentration= Moles of I2 * 176.12 g/mol * 25/100 mL
Moles of I2= 0.0000049 mol
Vitamin C concentration (g/100 mL)= 0.0000049 mol * 176.12 g/mol * 25/100 mL= 0.02157 g/100 mL
The purpose of this study is to observe the effects of the addition of the external cytokinin 6-Benzylaminopurine and MWCNTs on the growth rate of B. olceara. It was hypothesized that when both cytokinin 6-Benzylaminopurine and multiwalled carbon nanotubes (MWCNTs) were used in conjunction, these two factors would increase plant growth at an elevated rate compared to using them separately. This is justified through previous research which demonstrated that both cytokinin 6-Benzylaminopurine and multiwalled carbon nanotubes (MWCNTs) have been shown to increase plant growth and flowering rate individually. Additionally, increased plant growth is inherently tied to increased nutritional content in plants, resulting in the inclusion of this variable in the hypothesis. This hypothesis was tested through experimentation using the four media compositions and results were observed.
As observed in Graph 1, the 6-BAP group’s change in mass over time indicates that the plants were losing mass, which is not possible. These measurements are thus attributed to error with the scale and because of the difficulty in accurately weighing the miniscule microgreens.
The experimental group’s change in mass over time was the second highest in Trial 1, behind only the MS group, meaning that the conjunction of the 6-BAP and MWCNT did lead to a greater effect than those produced by the variables alone. But,as the 6-BAP inhibited growth in this trial, the MS group was higher than the experimental group in change in mass. If the 6-BAP worked as it did in previous literature, the experimental group would have had the leading change in mass over time. Due to errors previously delineated, this data does not demonstrate that the experimental group had the highest change in mass, but does show that the experimental group led to greater effects than those of the MWCNT and 6-BAP individually, as hypothesized.
In Graph 2, the change in mass data for trial 2 is present. In this trial, the Both (experimental) group had the third most change in mass, behind the 6-BAP and MWCNT groups. The results from this trial are exactly opposite of the results of trial 1, indicating that errors in growing conditions and mass measurements were most probably the cause for this disparity. The results of trial 2 do not corroborate the hypothesis as they demonstrate that the independent variables had a greater effect on growth rate individually in comparison to their effects together, and that the experimental group did not have the highest change in mass over time. As the trial 1 and 2 data do not demonstrate similar results, growing procedures and mass measurement methods need to be revised in future experimentation.
Additionally, as seen in Table 16, the p-values for the Fresh Weight and Dry Weight data seem to be < the established significance value of 0.05 (0.0141 and 0.0086 respectively), meaning that the differences between the control group’s fresh weights and dry weights and those of the experimental group are significant. This corroborates the hypothesis as it demonstrates that the experimental group has higher masses than the other groups. But, these significances are only due to error in measurement of control group fresh and dry weights, as demonstrated in Tables 1-6. During experimentation, many trials had seeds not growing and had mold in the jars, making the mass measurements of the extract plants negligible. As these minute masses were still recorded as data points of fresh and dry weight in the calculation of p-values, any slightly greater experimental group fresh or dry weight would have resulted in a significant difference from the extremely small, inconclusive fresh and dry weight measurements of some of the control groups in various trials. Thus, even though the p-values appear to depict a statistical significance, this is only due to errors in growing conditions or planting methods, and, in reality, both of these dependent variables are inconclusive in demonstrating a strong correlation between the experimental group and increased plant growth.
Table 14 depicts the anthocyanin concentrations of each of the five individual microgreens of each of the four media contents in Trial 3. It appears that the MWCNT group microgreens had the highest anthocyanin concentrations, with the MS media group microgreens in second, and the 6-BAP and Both group microgreens intertwined in terms of anthocyanin concentration differences. Surprisingly, plant 1 of the 6-BAP group had the highest anthocyanin concentration at 77.1526 [A]/g, indicating the variability in the masses of the 6-BAP group. However, since the anthocyanin concentration formula relies on dividing by the dry weight, plants with the lowest mass could be favored in anthocyanin concentration calculation, leading it to be the highest of all plants. But, plant 3 of the 6-BAP group had the lowest anthocyanin concentration at 10.21605 [A]/g, which highlights the variability in mass of the 6-BAP plants. This was most likely due to the usage of a slightly skewed concentration of 6-BAP in the media, which needs to be researched upon and fixed in future experimentation. As seen in Table 16, the p-value for the anthocyanin concentration is insignificant (0.1013), which further confirms that the anthocyanin concentration data is inconclusive in determining a definitive effect of the experimental group on nutritional content.
In Table 15, the Vitamin C concentrations of the four media content groups are depicted. Surprisingly, the experimental group had the least Vitamin C concentration (0.01431 g/100 mL) and the MS media group had the greatest Vitamin C concentration (0.02157). From all of the dependent variable measurements, it is reasonable to conclude that the MS media group is growing normally and has been consistently demonstrating higher amounts of all dependent variables. As for the experimental group, the lowest Vitamin C concentration again indicates that errors were present in the growing conditions or in the measurements of these dependent variables. The p-value depicted in Table 16 (0.99) for Vitamin C concentration is a very strong indication that is there is no statistically significant difference between the control and experimental groups in regard to this dependent variable.
Only the change in mass over time measurements in trial 1 seemed to somewhat corroborate the hypothesis that the experimental group would exhibit greater effects on plant growth and nutritional content than the independent variables alone. But, even these results were somewhat invalid as measurement error led to a decrease in mass of the 6-BAP group plants, and because the MS media group had a higher change in mass over time than any other group in Trial 1. All other data from the other dependent variables (change in mass over time (trial 2), fresh weight, dry weight, anthocyanin concentration, Vitamin C concentration) either had false significant p-values (due to mold and measurement error) or had insignificant p-values. This lack of support for this hypothesis can largely be attributed to error in 6-BAP concentration used in the trials and to irregularities in the air stream and growing conditions in the lab. Only the MS media seemed to be somewhat consistent in maintaining its dependent variable measurements, indicating that the 6-BAP and MWCNT concentrations could have been skewed. Overall, most of the data was inconclusive in establishing a strong causality between the experimental group and increased plant growth and nutritional content. There seems to be somewhat of a correlation due to the results of change in mass over time in Trial 1, but this needs to be expanded upon in future experimentation and through future dependent variable measurements. Also, growth conditions need to be standardized for future experimentation in order to limit mold contamination and the optimal 6-BAP concentration for B. oleracea microgreens should be researched.
In all the trials conducted, it was shown that there was no significant evidence found pertaining to the relationship between cytokinin and plant growth. In most of the data, it was shown to inhibit rather than promote growth. Thus, since cytokinin seemed to have no effect, its sister hormone auxin could be an independent variable of interest instead. Auxin is responsible for cell elongation as well as the promotion of flowering in plants . Further research could be done regarding the interaction between auxin and MWCNTs.
Furthermore, density could be added as a dependent variable in order to indicate an increase in growth. Experimentation showed that plants in the experimental group were often smaller, but still had a significant mass. In Table 4, the Both 1 group plants seemed to have high dry weights, though their actual size was smaller than the other plants of other groups. Also, in Table 2 and Table 6 (fresh and dry weight, respectively), though there were only two plants in the Both 1 group, their masses were some of the highest in their entire trial. Even though these plants were also small in stature, they seemed to be heavier. These data points indicate that density could be a viable dependent variable to indicate growth for future experimentation. This increased density could be attributed to the presence of MWCNTs or higher water content.
Finally, in order to determine whether MWCNTs actually entered the plant, new techniques such as Raman microscopy could be utilized to find the distribution of nanoparticles within the plants themselves. Acquiring a Raman microscope would be instrumental in determining the interactions between the plants and the MWCNTs, and could help in the construction of better methods of delivery of MWCNTs to the microgreens.
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