The response of Dundubia Manifera sound effects to changes in stomata density and stomata index of water spinach as information on the rate of photosynthesis

. Sound waves are vibrations that propagate through a medium. Sound waves can be applied to agricultural land as a technology that is often called Plant Acoustic Frequency Technology (PAFT). This technology is applied to agricultural land by using sound that has been manipulated with a certain frequency. This study aims to provide the sound effect of Dundubia Manifera to determine the effect on stomatal density and stomatal index. This sound has been manipulated using Adobe Audition CS6 to be 4000 Hz which will be applied 30 minutes for 28 days. The results show that stomatal density and stomata index are affected by exposure to sound waves. In which, stomatal density was reduced by 89% with control plants (97,506 /mm2) and treatment plants (51,428 /mm2). Meanwhile, the stomatal index increased by 41% with control plants (0.194 m) and treatment plants (0.274 m). These results indicate that stomata have a very good impact on plants such as increasing the rate of photosynthesis, plant immune systems, surviving climate change, and increasing crop yields.


Introduction
Climate change that often occurs today, such as global warming and changes in rainfall, will have a negative impact on agricultural ecosystems.This will affect the production of food crops such as wheat, rice, corn and other crops [1].In addition, the high market demand for food crops needs to be followed up to increase the amount of production.Another problem that often arises is that plants are susceptible to disease during climate change.Climate change can trigger plants to become infected with pathogens because the agricultural environment is not conducive [2,3].Thus, it is necessary for researchers to make efforts to increase agricultural production and improve plant immune systems to survive rapid environmental changes [4].One of the techniques used to deal with the challenges of climate change is to apply sound waves with a certain frequency to plants.This technique will promote growth, increase production, improve quality and increase plant resistance to disease as well as plants that are able to withstand drought [5].Sound-induced vibrations can contribute to the plant's chemical defense system [6].
Plant Acoustic Frequency Technology (PAFT) in plants has been studied for decades.Many studies and experiments have been carried out by researchers to determine the effective sound frequency on several types of plants.Based on a study conducted that PAFT with frequency, sound pressure levels (SPLs), exposure period and plant distance with different sound sources will affect plant growth [7].This is because plants have different growth stages.Some researchers have tried to use several sound sources with different sound frequencies and intensities such as animal sounds [8] and music (classic, rock and others) [9].In general, plant response is optimal by providing a frequency of 3-5 kHz [9,10].This is because at this frequency the stomata opening is wider so that the absorption of plant nutrients through the leaf stomata is more optimal [11].
The growth of water spinach (Ipomea Aquatica) is closely related to the effectiveness of stomatal openings.Stomata are an important part of plants that play a role in the exchange of CO2, oxygen, gases and other compounds through the pores of the epidermis so that it affects the rate of photosynthesis [12,13].Stomata can control water loss when transpiration and CO2 exchange during the photosynthesis process.Large stomata size can maintain the rate of photosynthesis for plants that have excess CO2 and decrease water [14].The rate of photosynthesis can be studied by determining the stomatal density and stomatal index through observation.Stomata density or stomata index is an important factor for plants to maintain growth from water loss to continue to produce optimal photosynthesis.[4].
In this study, sound waves can theoretically increase the rate of photosynthesis.Proof of this theory can be obtained by determining the stomatal density and stomatal index through measurements using a microscope to determine the stomatal density, epidermal cell, and field of view [15,16].

Materials
The experiment was conducted from August to September 2021.The tools and materials used in this study were water spinach (ipomea aquatica), plastic bags, soil, fertilizer, scissors, ruler, analytical guide, sound level meter, PAFT device and microscope.

Experimental treatment
Pots containing water spinach were randomly designed using two conditions (treatment and control).There are 25 pots of plants for each group in one area.The environment was in the same condition by ensuring the distance between the treatment and control plants were close together.The application of sound waves is carried out in the box, so that homogeneous control plants are still inserted into the box.When performing sound wave treatment, the two boxes are separated by 5 m to reduce noise other than the sound from the PAFT device [17].The sound used is the voice of Dundubia Manifera with an exposure duration of 30 minutes.This application is carried out for 28 days until the harvest.

Determine stomatal density and stomata index
After harvesting, the treatment and control plants were selected by selecting five plants that had very good quality in terms of size.These five plants will be measured stomatal density and stomata index to determine the difference.The procedure for observing leaf stomata can be done by giving a longitudinal incision on the lower surface of the water spinach, then placing it on a prepared glass.Add a drop of distilled water over the incision and cover with a cover slip.Next, observe using a microscope from a low magnification of 10x.Measure the length and width of the stomata using a microscope on the eyepiece of the microscope.Observe the number of stomata visible in the field of view under the microscope.The results of this measurement are used to determine the stomatal density using formula (1) [16]: Meanwhile, to determine the stomatal index using formula (2).Where, the Stomata Index Formula is,

Result and discussion
Crops field can be optimized by applying sound waves using certain frequencies.Water spinach became the object of research because it has a short harvest time of 28 days and has the convenience of analyzing leaf stomata.The results of the observation of five plant samples, both control (A) and treatment (B) can be seen in Figure 1.In this investigation, the technique used to determine stomatal density and stomatal index can be determined through observation under a microscope by determining the number of stomata as visible in the circular view field of the microscope (Fig. 1).After making observations in the laboratory, then comparing the results of stomatal density and stomata index of control (A) and treatment (B) plants which can be seen in Figures 2 and 3  Several researchers continue to strive to optimize plant growth by modifying plants to reducing the number of stomata so as to increase drought tolerance without reducing crop yields [1].The results of this study showed that the response of sound waves in the treatment plants had a decreasing trend.This occurs because the number of stomata decreases and the size of the stomata is wider.Based on the statistical test using the t-test, the treatment plants did not show significant results at p=5% because the difference in values was small.Even so, graphic observations show changes in the value of stomatal density and affect the number of stomata.Application of sound with various frequencies can contribute to drought tolerance which increases water content, stomatal conductance and quantum yield in drought stress environments.[18].In addition, plants with large stomata and small numbers can maintain the rate of photosynthesis.This is very useful when increasing CO2 and decreasing the availability of water so that plants remain optimal in the dry season [14].Supported by research [19] that rice plants with low stomatal density can be able to maintain stomatal conductance conditions and last long enough at a temperature of 40°C because they can overcome the problem of water drought and in the future can work in climate change that threatens food security.In addition to stomata density which is an important factor for plant growth, the stomata index needs to be calculated as additional information to show an increase in the rate of photosynthesis.Figure 3 shows the data for calculating the stomata index of control plants and treatment plants.The results showed a higher stomatal index due to exposure to sound waves.Supported by t-test statistical analysis, it was found that the stomata index gave significant results at p=5%.This means that there is a significant change in the stomatal index after exposure to sound waves for 28 days before harvest.Based on the research of Segev et al [4] who found a significant correlation between the stomata index and photosynthesis.A higher stomata index will affect the rate of photosynthesis even in a dry environment.
From the data obtained, sound waves are the main factor in changing the characteristics of stomata in leaves.The effect of 4 kHz sound in plants can cause the stomata to open wider.4 kHz sound effect can also increase drought tolerance, yield increased by 40.89% and soybean protein content increased by 10.3% [20].Other benefits of applying sound waves to plants can increase plant resistance to disease attacks, reduce the use of chemical fertilizers [21] and are able to deal with extreme climate change.Sound waves with specific frequency and intensity can have a positive effect on germination, root elongation, plant height, callus growth, cell cycling, signalling transduction system, enzymatic and hormonal activities and gene expression [22].

Conclusion
Exposure to sound waves with a frequency of 4000 Hz has a great impact on plant growth.The density of small stomata with wide stomata opening causes the process of photosynthesis through the stomatal pores to be more optimal.In addition, the results of a large stomatal index have a very good impact on the rate of photosynthesis.That is, exposure to sound waves can have an effect on plant physiology so that it affects the rate of photosynthesis and increases the immune system in surviving in a dry environment and bacterial attacks that interfere with plant growth.

Fig. 1 .
Fig. 1.Microscopic view of stomata observation of water spinach with 10x magnification where symbol A = control plant and symbol B = treatment plant. .

Fig. 2 .
Fig. 2.Comparison of stomatal density data of water spinach plants given exposure to sound waves (treatment) and without treatment (control).The results of the statistical analysis t-test of stomatal density there was no significant difference at p=5%.

Fig. 3 .
Fig.3.comparison of the stomatal index of water spinach plants given exposure to sound waves (treatment) and without treatment (control).The results of the statistical analysis test t-test stomatal index there is a significant difference at p = 5%.
This research is supported by LPPM University of Bengkulu through the 2021 Fundamental scheme with the contract number: 1810/UN30.15/PG/2021.Thanks also to the Science Laboratory D3 Study Program which has facilitated the use of the Laboratory.