Root to Fruit: Polyphenol Oxidase Regulation in Plants to Improve Food Security.

Pear Orchard - Cherry Tree Farm

Beamsville Ontario.

Caption:

Hi My Name, is Avi Joshi and I am a Grade 8 student.

Post-harvest Agricultural Produce is frequently compromised on farms, during transportation from farms to warehouses and retail stores. Spoiled produce is subsequently thrown out from grocery stores into landfills, where the waste anaerobically decomposes, thereby emitting harmful greenhouse gases such as Methane.  Did you know a shocking 644 million Tons of crops are wasted annually.

The majority of this wastage in produce is primarily caused by Enzymatic Oxidation. The Polyphenol Oxidase Enzyme is responsible for causing this Enzymatic Oxidation by acting as a Biological Catalyst when exposed towards Oxygen.  In this experiment, the NCBI Databank was used to identify the Polyphenol Oxidase Gene. I then edited the Polyphenol Oxidase Gene using CRISPR/Cas-9 Mediated Gene Editing in somatic embryo cultures of the species Vitis Vinifera. This disrupted the Polyphenol Oxidase Gene.  Finally, to confirm the edit was successful, a Chemical Assay was performed that quantified the lowered concentration of PPO Enzymes.

Thus, managing PPO levels in fresh produce can be one way to reduce food wastage, improve food security and meet the global demand for proper quality food.

Despite multiple advances in agriculture to improve food production, the availability of food for the growing global population remains a serious problem. Approximately more than 644 million tonnes of all crops produced end up being wasted, over 40% of Fruits and vegetables are wasted every year (Hegnsholt et al., 2018).

Human population is rapidly rising, and numerous consumers have insufficient nutrition due to limited access to produce. This makes it very necessary for us to discover new technologies by which we can overcome these adversities and produce a greater amount of food and provide sustainable agriculture to supply the rising human population.

Produce wastage especially for fresh produce occurs when fruits and vegetables become damaged during harvesting, shipping and storage subsequently turning brown in color and losing their nutritional value and marketability. Food browning is caused by oxidation of damaged tissues by an enzyme called polyphenol oxidase (PPO).

If the PPO levels can be efficiently managed with reduced Enzymatic Oxidation, we can decrease the amount of food wastage after harvesting, and help improve, availability and security. The overall shelf life and nutritional content can be preserved as well. This also allows the fruit or vegetable to have a proper defense mechanism as well as also have an increased marketability and reduced wastage.

Thus, managing PPO levels in fresh produce can be one way to reduce food wastage, improve food availability and security, and meet the global demand for proper quality food.

Observation: To develop my understanding of Polyphenol Oxidation process, I collected commonly available fruits and vegetables from local grocery stores. Produce were cut at the same time and were subsequently observed for Enzymatic Oxidation Activity for 1-48 hours.

Experiment Procedure:

Step 1: Use the NCBI (National Center for Biotechnology Information) Data Tools to obtain DNA and Protein Sequences

Step 2: Compare these Sequences with another Data Chart and see the similar homology in the BLAST Tool.

Step 3: Grow the Agrobacterium Plasmids containing CRISPR/Cas-9 construct for 12 hours in shaking incubator in liquid bacterial culture medium.

Step 4: Separate Cell Cultures to have embryos at a low maturity stage to avoid SEG (Secondary Embryo Genesis) for Higher chance of results of infections.

Step 5: Mix grape cell cultures and the bacterial culture in a Petri dish while taking care bacterial culture so it does not spill out of the dish.

Step 6: Let the mixture of bacterial culture and grape cell culture incubate for 10 minutes and then remove all the bacterial culture.

Step 8: After 2 days, move the grape cell cultures from the petri dish to a flask containing liquid growth medium and antibiotics to kill the Agrobacterium.

Step 9: Wash cells for one day and then transfer to solid tissue culture medium in the dark for 30 days.

Step 10: When the embryos develop leaves and are sprouting collect the leaves and grind them as well as adding 2 buffers into the grind.

Step 11: After the grind filter out the liquid and place into small tubes. While then transporting the tubes into the Spectrophotometer.

Step 12: Once the tubes are in the spectrophotometer run the machine to obtain the results.

Step 13: Collect the samples and then record the results, then make a graph.

The edited cells need to be confirmed for reduced PPO Enzyme Activity. The Chemical Assay was performed in order to obtain the results of the PPO Enzyme concentration. The Chemical Assay uses a machine known as the Spectrophotometer to use wavelengths at 420 NM to analyze the data and presents the findings in the form of a table using a software. The 30 separate leave samples that were edited and then analyzed for PPO Enzyme Concentration data. The results shows that only 4/30 samples had actually reached the optimal quantity of PPO Enzyme Concentration when exposed towards Oxygen. This was mainly due to the fact that contamination or increase of maturity of the cells, during the gene editing procedure could have altered the results, causing majority of the edits to be unsuccessful.

The reason why this process is extremely important is because it confirms whether the gene edit was actually successful or not and can determine whether or not to cultivate the edited cells and grow them into commercialized crops to then be utilized for sustainable methods of agricultural production. Optimal Quantity of PPO Concentration is better when traces are still found instead of no traces of PPO Enzymes. This is because it allows the fruit to keep a defense mechanism used against parasites while still being able to be commercially sold, these techniques satisfies these two requirements in order to provide sustainable agriculture hence why it was chosen.

In conclusion, we applied how Biotechnology tools such as Bioinformatics and Genetic Engineering via CRISPR/Cas-9 Mediated Gene Editing, works in commercial uses for plants to identify, sequence and edit DNA sequences. These techniques were used to the disrupt the Polyphenol Oxidase gene in the genome of the Common Grape Vine (Vitis vinifera). This helps develop fruits and vegetables with reduced browning

Databanks such as the NCBI provide useful tools/information of identified gene sequences.

Technologies like CRISPR/Cas-9 can help us improve the varieties of crops we grow for different characteristics such as reduced enzymatic browning and higher nutritional content. If we improve characteristics in crops, we can grow our crops in an eco-friendly manner and we can protect our environment while ensuring we can grow produce with enough nutritional content and longer shelf-life to feed the growing global population.

This experiment is a commercial use of Biotechnology tools like CRISPR/Cas9. This technique can also be used to have more than 1 edit also known as Multiplexing. The resulting crop will have an improved resistance to diseases resulting in less usage of pesticides as well as decreasing the browning of crops and reducing food wastage.

Thus, modern tools in Biotechnology can revolutionize agriculture and help us produce more food for an increasing population in a more sustainable and Eco-friendly manner.

Future applications involving the current techniques used in this experiment, specifically CRISPR/Cas-9 Mediated Gene Editing, can be used for more than 1 characteristic trait in crops. This technique is known as Multiplexing, where you genetically disrupt more than 1 trait in crops. For example, targeting a gene involved in the production of a protein that pathogens use to identify whether the targeted plant is suitable for infection and targeting the PPO Gene Involved in Polyphenol Oxidase enzyme production. Both of these edits will result in an increase in sustainable methods to supply agriculture for the growing population, utilizing Biotechnology.

Firstly, I would like to thank my parents for helping me find a lab and supporting my interests in Biotechnology and Molecular Biotechnology.

I would also like to thank my teachers and the NRSEF team for showing how CWSF works and also informing me about strategies as well as preparing me for it for CWSF.

Then I would also like to BIG thank you to Dr. Sadanand Dhekney for providing me a lab to conduct the experiment as well as Gavin Souter for mentoring me about how to perform the techniques used.

Finally I would like to thank Dr. Purushothaman Natarajan for teaching me about Bioinformatics and useful tools such as Clustal omega.

Ahmad, Aftab, et al. “GMOs or Non-GMOs? The CRISPR Conundrum.” Frontiers in Plant Science, vol. 14, Frontiers Media SA, Oct. 2023, https://doi.org/10.3389/fpls.2023.1232938. Accessed 29 Apr. 2026.

‌‌

Korycki, Lisa. “Moving the Dial to End Food Waste.” Waste Management Review, 27 June 2024, https://wastemanagementreview.com.au/moving-the-dial-to-end-food-waste/. Accessed 29 Apr. 2026.

Ottaviani, Javier I, et al. “Impact of Polyphenol Oxidase on the Bioavailability of Flavan-3-Ols in Fruit Smoothies: A Controlled, Single Blinded, Cross-over Study.” Food & Function, vol. 14, no. 18, 1 Jan. 2023, pp. 8217–8228,

Esben Hegnsholt, et al. “Tackling the 1.6-Billion-Ton Food Loss and Waste Crisis.” BCG Global, 20 Aug. 2018, https://www.bcg.com/publications/2018/tackling-1.6-billion-ton-food-loss-and-waste-crisis. Accessed 1 May 2026.

Sheets, Mariel. “Apples of the Future: The Argument for Genetically Modified Apples - The So-Called.” Schmid College of Science and Technology, 2016, https://blogs.chapman.edu/scst/2015/11/23/apples-of-the-future-the-argument-for-genetically-modified-apples/. Accessed 1 May 2026.

https://www.facebook.com/sag.micro. “Spectrophotometer: Principle, Instrumentation, Applications.” Microbe Notes, May 2024, https://microbenotes.com/spectrophotometer-principle-instrumentation-applications/. Accessed 1 May 2026.

pubmed.ncbi.nlm.nih.gov/37615673/#:~:text=The%20peak%20plasma%20concentration%20(C,home%20and%20in%20industrial%20settings., https://doi.org/10.1039/d3fo01599h. Accessed 8 Nov. 2025.

Dhekney, S.A., S.K. Sessions, M. Brungart-Rosenberg, C. Claflin, Z.T. Li and D.J. Gray 2019.Genetic modification of grapevine embryogenic cultures. In: Kumar, S., Barone, P., and Smith, M. eds. Transgenic plants: methods and protocols, Springer Publications, pp 191-201

Glagoleva, Anastasiia Y., et al. “Melanin Pigment in Plants: Current Knowledge and Future Perspectives.” Frontiers in Plant Science, vol. 11, 23 June 2020, www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2020.00770/full, https://doi.org/10.3389/fpls.2020.00770. Accessed 4 Oct. 2025.

B, KAYLA. “The Problem With Food Waste on a Grand Scale.” Ally’s Kitchen, 3 Sept. 2024, https://allyskitchen.com/the-problem-with-food-waste-on-a-grand-scale/. Accessed 1 May 2026.

Maioli, Alex, et al. “Simultaneous CRISPR/Cas-9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum Melongena L.” Frontiers in Plant Science, vol. 11, 3 Dec. 2020, pmc.ncbi.nlm.nih.gov/articles/PMC7744776/, https://doi.org/10.3389/fpls.2020.607161. Accessed 4 Oct. 2025.

‌

Qin, Fei, et al. “Genome-Wide Analysis of the Polyphenol Oxidase Gene Family Reveals That MaPPO1 and MaPPO6 Are the Main Contributors to Fruit Browning in Musa Acuminate.” Frontiers in Plant Science, vol. 14, 14 Feb. 2023, www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1125375/full, https://doi.org/10.3389/fpls.2023.1125375. Accessed 28 Nov. 2025.

Yang, Shuhan, et al. “Genome-Wide Identification and Characterization of the PPO Gene Family in Cotton (Gossypium) and Their Expression Variations Responding to Verticillium Wilt Infection.” Genes, vol. 14, no. 2, 13 Feb. 2023, p. 477, pmc.ncbi.nlm.nih.gov/articles/PMC9957175/#abstract1, https://doi.org/10.3390/genes14020477. Accessed 4 Dec. 2025.

Maioli, Alex, et al. “Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum Melongena L.” Frontiers in Plant Science, vol. 11, 3 Dec. 2020, www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2020.607161/full, https://doi.org/10.3389/fpls.2020.607161. Accessed 1 Oct. 2025.

González, Matías Nicolás, et al. “Reduced Enzymatic Browning in Potato Tubers by Specific Editing of a Polyphenol Oxidase Gene via Ribonucleoprotein Complexes Delivery of the CRISPR/Cas9 System.” Frontiers in Plant Science, vol. 10, 9 Jan. 2020, www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2019.01649/full, https://doi.org/10.3389/fpls.2019.01649. Accessed 7 Nov. 2025.‌

Toro-Uribe, Said, et al. “Insight of Polyphenol Oxidase Enzyme Inhibition and Total Polyphenol Recovery from Cocoa Beans.” Antioxidants, vol. 9, no. 6, 27 May 2020, p. 458, pmc.ncbi.nlm.nih.gov/articles/PMC7346217/#:~:text=Precursors%20of%20chocolate%20flavor%20are,and%20quality%20value%20%5B13%5D., https://doi.org/10.3390/antiox9060458. Accessed 14 Nov. 2025.‌