Vigna radiata, commonly known as mung beans, hold significant agricultural importance in Kenya, much like in many other regions globally. "In Kenya and Tanzania, these legumes are cultivated on approximately 302,292 and 148,885 hectares, respectively. They are prized for their rich protein content, measuring at \(23-25\%\), alongside valuable carbohydrates and dietary fibres." (Mmbando & Mbeyagala, 2021) Apart from being a staple crop, mung beans offer income opportunities for farmers and serve as a vital source of nutritious food. "Notably, they thrive in Kenya's eastern and western regions, where they yield about 42,321 tons per hectare as of 2019, thereby contributing to food security." (Mwangi et al., 2021) Furthermore, mung beans serve as an affordable protein alternative for those unable to access animal protein. Their water-efficient and low greenhouse gas emission characteristics make them a sustainable choice, standing out even among other plant-based protein sources like soy. (Lazimy, 2020) Growing up in Kenya, mung beans have been a staple in my diet, featured in dishes at home at least once a week. My parents own a small farm in Malindi on the coast of Kenya, growing various crops, and were considering growing mung beans due to our frequent consumption at home. However, we were uncertain about the optimal planting conditions for these legumes. Hydrogen peroxide can be used to maximise yield as it increases germination rate, many farmers are unaware about the benefits of using hydrogen peroxide in increasing crop yield. To address this, I aim to investigate the effects of various hydrogen peroxide concentrations on Vigna radiata germination. As hydrogen peroxide is a common solution to increase germination speed. By identifying the optimal conditions for growth, the study can contribute to increased economic growth in the agricultural industry, benefiting both farmers and consumers alike.

Seed germination is an important process in the life cycle of plants. It marks the growth of a new plant from a seed. This process begins when a seed, usually in a state of dormancy, absorbs water and swells. At the same time, metabolic and physiological processes within the seed occur, leading to the emergence of the radicle (embryonic root and the shoot), developing into a seedling; this process is known as seed germination. The success of seed germination is vital for plant propagation, crop production, and the persistence of plant species in natural ecosystems. (Heslop, 2023)

Several factors need to be met for the germination process to take place. First, the embryo must absorb water in order to grow its cells and rehydrate the tissues of the dry seed. For aerobic respiration to produce the ATP required for growth, oxygen is required. Last but not least, the seed's enzymatic activity must be optimised at a certain temperature. The seed will start to germinate soon after all of these requirements are met. (Sghaier et al., 2022) Temperature plays a pivotal role, with different plant species having specific temperature preferences, with warm-season plants favouring higher temperatures and cool-season plants thriving in lower temperatures. Light can be either a requirement or an inhibitor, varying by plant species, with some needing light exposure for germination while others preferring darkness. (Sghaier et al., 2022)

To increase the rate of germination, seeds can be soaked in hydrogen peroxide - a chemical compound with the formula \(\mathrm{H}_{2} \mathrm{O}_{2}\). It is chemically similar to water \(\mathrm{H}_{2} \mathrm{O}\) the main difference being the additional oxygen atom. Hence why hydrogen peroxide can play a crucial role in seed germination as it softens the seed coat allowing the seeds to absorb more oxygen in. Resulting in an increased germination rate. Additionally, it lacks harmful additives and is non-toxic, environmentally friendly, and fully biodegradable into oxygen and water, making it a safe choice for plants. (Speed Up Seed Germination Using Hydrogen Peroxide, n.d.)

Literature documenting hydrogen peroxide use as a seed treatment for V.radiata is lacking, however similar studies found that hydrogen peroxide promotes seed germination in other plant species by dissolving the hard seed coat.(BENDER, n.d.) According to a literature review on the role of hydrogen peroxide \(\left(\mathrm{H}_{2} \mathrm{O}_{2}\right)\) in pea seed germination. "Hydrogen peroxide influences pea seed germination positively, with concentrations up to 20 mM enhancing both germination and radicle growth. However, higher concentrations ( \(>20 \mathrm{mM}\) ) stimulate germination initially but lead to abnormal radicle growth later. Concentrations exceeding 100 mM reduce germination rates. Notably, the timing of \(\mathrm{H}_{2} \mathrm{O}_{2}\) application matters: \(5 \mathrm{mM} \mathrm{H}_{2} \mathrm{O}_{2}\) during incubation yields similar germination rates as control seeds, while 10 or \(20 \mathrm{mM} \mathrm{H}_{2} \mathrm{O}_{2}\) negatively affects germination when applied post-imbibition." (Barba-Espín et al., 2012) Although the literature review focuses on pea seeds, a study on the effects of \(\mathrm{H}_{2} \mathrm{O}_{2}\) on photosynthesis and polyphenolic compounds in mung beans found that \(\mathrm{H}_{2} \mathrm{O}_{2}\) consistently damaged plants and affected their activities. (Science Direct, 2023) However, plants with minor damage recovered their photosynthetic activities and growth. Another study on the role of \(\mathrm{H}_{2} \mathrm{O}_{2}\) and cell wall monoamine oxidases in germination of Vigna radiata seeds found that \(\mathrm{H}_{2} \mathrm{O}_{2}\) generated within cell walls of seeds serves as a signalling molecule guiding germination events, including protein reserve mobilisation. (Sharma, 2010)

The increase in concentration of hydrogen peroxide solution does not affect seed germination, as measured by the radicle length (mm) of germinating Vigna radiata seeds after 72 hours.

As the concentration of hydrogen peroxide solution increases when germinating V.radiata seeds, the rate of germination measured by the radicle length ( mm ) undergoing germination after 72 hours will also increase. Lower concentrations of hydrogen peroxide may promote and enhance germination due to their potential role in promoting germination, while higher concentrations of hydrogen peroxide may hinder or inhibit germination

due to oxidative stress. As seen in figure 1, a study reported when \(5 \mathrm{mM} \mathrm{H} \mathrm{H}_{2} \mathrm{O}_{2}\) was introduced during the incubation process following seed imbibition in \(\mathrm{dH}_{2} \mathrm{O}\), the percentage of germinated seeds closely resembled that of the control group. Conversely, the inclusion of 10 or \(20 \mathrm{mM} \mathrm{H}_{2} \mathrm{O}_{2}\) in the Petri dishes led to an adverse impact on seed germination. (Barba-Espín et al., 2012) Therefore, it is expected to observe a concentration-dependent response, with some concentrations yielding higher germination rates compared to others. (Wojtyla et al., 2016) Additionally as the concentration of hydrogen peroxide reaches higher concentrations the acidity of the solution may affect the seeds and decrease the germination rate. A high concentration of the hydrogen peroxide can make the seed coat tissues degrade and cause seed/embryo damage. (Gentili et al., 2018) However these studies may be focused on germination rates and not germination alone they help in drawing a hypothesis in relation to the effect of \(\mathrm{H}_{2} \mathrm{O}_{2}\) concentrations on V.radiata seed germination.

A preliminary trial was conducted to establish the ideal germination duration for Vigna radiata seeds. Originally, 24 or 48 hours were considered for germination to gather sufficient data. However, it became evident that the seeds did not exhibit significant growth within these time frames. As a result, a 72-hour germination period was deemed appropriate to provide the necessary data to validate the hypothesis effectively.

During the preliminary trials, a range of hydrogen peroxide concentrations, from \(0\%\) to \(5\%\), were experimented with. It was observed that the seeds exposed to higher concentrations, specifically those between \(3\%\) and \(5\%\), did not germinate successfully. Consequently, the concentration range was narrowed down to \(0\%, 0.5\%, 1\%, 1.5\%\), \(2\%\), and \(2.5\%\). This selection was made to identify the optimal hydrogen peroxide concentration that would yield the highest germination rates.

Independent variable:
> Concentration of Hydrogen Peroxide solution- the concentration of Hydrogen Peroxide solution when Vigna radiata seeds were soaked in it was the independent variable as it changed increasingly from \(0.5\%, 1\%, 1.5\%, 2\%\) and \(2.5\%\). The concentrations were prepared right before soaking the seeds as Hydrogen Peroxide tends to decompose. The preparation was done by using the following equation as only \(6\%\) hyrogen peroxide was available at the lab.

\(\frac{\text{concentration needed} \times 100}{6}=\) volume of \(\mathrm{H}_{2} \mathrm{O}_{2}\) to add to 100 ml water

The above equation was used to determine the volume of hydrogen peroxide needed per 100 ml of water for the desired concentration.

Dependent variable:
> Length of seed radicle grown in 72 hours- the length of the Vigna radiata seed radicle grown in millimetres ( mm ) is the dependent variable. This was measured using a ruler from the hypocotyls to the tip of the radicle.

1. Prepare six beakers, each containing 50 ml of hydrogen peroxide solution with a different concentration: \(0.0\%, 0.5\%, 1.0\%, 1.5\%, 2.0\%\), and \(2.5\%\).

2. Place 10 mung bean seeds in each of these beakers and allow them to soak for 1 hour.

3. Label each petri dish with the corresponding hydrogen peroxide concentration (\(0.0\%\), \(0.5\%, 1.0\%, 1.5\%, 2.0\%\), and \(2.5\%\)) and trials 1-5.

4. In each labelled petri dish, position two cotton pads approximately 1 cm thick and evenly distribute \(10\mathrm{cm}^3\) of the respective hydrogen peroxide concentrations onto the cotton pads.

5. Using forceps, place 10 seeds in each petri dish according to the concentrations they were soaked in and leave for 72 hours.

6. After 72 hours, carefully remove each seed from the petri dish and measure the radicle length on a flat surface.

a. Ensure that the measurement is taken from the hypocotyls to the tip of the radicle.

7. Repeat the same measurement procedure for each seedling in every trial and concentration.

Safety:
- Wear personal protective equipment such as gloves, goggles and lab coat when handling hydrogen peroxide as it can be corrosive and cause skin and eye irritation. Handle hydrogen peroxide with care, as it is a strong oxidising agent and can be hazardous if ingested or inhaled. (The President and Fellows of Harvard College, 2020)

Ethics:
- As the mung beans are a common food supply and in this case 300 seeds are being used just for the main trials of the experiment. This use can be seen as wasteful as the beans are not ingested and are used for experimentation. To avoid this waste the seedlings were planted in the school garden to ensure their potential for growth and utilisation beyond the experiment.

Environment:
- Given that high concentrations of hydrogen peroxide can have harmful effects on the environment, it is crucial to handle and dispose of it responsibly. Instead of directly releasing it into the environment, diluting the solution and safely disposing of it down the drain can help minimise potential environmental harm.

- When soaked in the hydrogen peroxide solution a few of the seeds started floating at the top while the rest remained at the bottom showing that some of the seeds had become less dense due to the soaking process, indicating changes in their internal structure or water absorption capacities. Most of the seeds that began to float in the solutions were in the \(2.50\%\) solution suggesting that these seeds absorbed a lot more water than the seeds in the other solutions.

- Additionally, many of the seeds in the \(2.00\%\) and \(2.50\%\) solution started growing a reddish-purple radicle instead of a white one, indicating unhealthy seedling growth as a healthy seedling would have a white radicle. These seedlings also had short and stubby radicles, which meant that they likely experienced inhibited root development compared to healthier seedlings with longer and slender radicles.