The relationship between various electric current voltage levels and the germination and post-germination growth of radish (Raphanus sativus L.)

How do different electric current voltage levels (0V, 3V, 6V, 9V, 12V) influence germination and post-germination growth of Raphanus sativus L.?

Contents

1. Introduction
2. Background information
2.1 Germination and post-germination plant growth.
2.2 Effect of electric current on plant growth
2.3 Characteristics of radish (Raphanus sativus L.)
3. Preliminary experiment
4. Variables
5. Hypothesis
6. Methodology
6.1 Equipment
6.2 Procedure
7. Results
8. Statistical analysis
9. Data analysis
10. Discussion
11. Conclusion
Bibliography

Abstract

The aim of the following investigation was to determine the effect electric current has on post-germination growth of Raphanus sativus L. (radish) seedlings. Such research can prove itself important for future agriculture, which instead of fertilizers, or with a lower amount, could use electricity as a stimulant for plant growth. The hypothesis was: application of electric current to seeds and seedlings of Raphanus sativus L. will result in accelerated growth (with optimum voltage) or in decelerated growth (with too low or too high voltage) of roots and stems. Specific voltages (0V, 3V, 6V, 9V, 12V) were chosen in order to provide a wide spectrum of data and facilitate the localization of voltage for which the change in growth was the greatest. The experiment involved 40 samples (8 per trial) and lasted 10 days. After that, the roots, stems, and total lengths of samples were measured. There was no correlation between length of stems or total length and the voltage applied to seeds. However, in the case of root length, the statistical test showed a significant difference between 0V and 3V trials. The difference corresponded to the root length increase of 40%. Nevertheless the research is inconclusive and further experiments need to be conducted in order to refute or support the hypothesis.

1. Introduction

Nowadays, alternative methods of fertilization in agriculture are searched for. Chemical substances like herbicides and insecticides lead to contamination of water and soil and by that to death of marine and land organisms². An alternative way of increasing crop yields, electroculture, was first investigated in the 18th century by Dr. Maimbray of Edinburgh³⁸. It involved use of electric current, and has since been subject to many experiments. Their results were often contradictory¹⁴ but it seems that due to the ubiquity of electricity⁹ in the natural environment, it should have an impact on living organisms. It may manifest itself in the orientation of cell division plates⁴¹ or in faster germination¹². Impact of electricity may be especially important in the uptake of minerals by roots and in phloem loading as their mechanism relies on the active transport with H+ mediatory molecules³. As the ions are affected by electric field their absorption should be affected too. Therefore, the growth of roots depending on the minerals in soil should be altered as well. That is an interesting possible outcome which shall be investigated in this paper. It will be focused on the influence of electric current on the germination time, measured by a number of seeds germinated each day and post-germination growth of Raphanus sativus L., assessed by the length of roots and stems of the seedlings. Therefore the research question of this paper is: how do various electric current voltage levels (0V, 3V, 6V, 9V, 12V) influence germination and post-germination growth of Raphanus sativus L.?

2. Background information

2.1 Germination and post-germination plant growth.

The first stage of plant growth is germination, which is the effect of absorption of water by the seed. The water molecules are attracted and bonded by the polysaccharides, large molecules made of simple sugars, present in the seed³⁰. These sugars can be further used in cell respiration¹¹. This process may be enhanced by applying the electric field, which causes water accumulation in cells¹⁸. Water plays a crucial role in plant growth as its accumulation exerts pressure on cellulose cell walls, leading to elongation of cells, especially after cell division³⁹. The cell walls have to be previously made flexible by the H+ ions, which are released as the result of action of auxins, a type of plant hormone relevant for plant growth¹¹. Auxin also has the ability to stimulate differential growth in response to gravity or light stimuli⁴². The tissue responsible for the increase in the size of plants and development of other tissues is the meristem⁸.

2.2 Effect of electric current on plant growth

According to Briggs et al. all plants are surrounded by electric current, as in the atmosphere the electric field strength is (on average) 100 Vm-1 ⁹. Rezaei-Zarchi et al. stated that electricity has an impact on plant growth indirectly, by affecting ion movement in the soil or directly, by affecting the electron transport chain and permeability of membranes²⁸. Similar observations were made by Isobe et al. i.e. the electric polarization of the membranes causes water accumulation¹⁸. It affects the plant growth hormone auxin, causes acceleration in the movement of nitrogen-containing substances from the endosperm into the roots and stems, and an accumulation of amino acids, nucleic acids and sugars¹². It was observed that the organisation of meristematic tissue in roots is affected by auxins and when the electric current is applied the organization of root meristem is disturbed³⁶. Ellis and Turner also reported that after treating cherry seeds with an electric current there was an acceleration of germination and an increased rate of breakdown of reserved substances¹². Another observation shows that each plant has its own electric potential levels in minimum, optimum and maximum⁶, and also that these levels vary with species and variety of plant⁷. McKinley found that 1 minute exposures to electric field caused retarded germination, and 30 to 40 second exposure caused accelerated growth of corn seedlings during the early germination period²³. Unlike conventional transplanters, this vegetable planter does not require handling of seedlings during the transplanting process. A similar experiment conducted by Stone on radish showed the increase by 28.5% in weight in plants treated with electric current³³. He used two electrodes placed opposite to one another in soil, resulting in a small electric current. This method was used in the experiment for this paper.

2.3 Characteristics of radish (Raphanus sativus L.)

Radish is a cool-season root vegetable and a member of the Cruciferae (mustard family)⁴. Radish is widely chosen as a research plant, for example in studies of Ivánovics and Horváth, and Craker, Seibert and Clifford^19,10. The reason for this is its rapid growth²⁴ and low cultivation requirements (e.g. growth in a wide range of pH²¹).

3. Preliminary experiment

Before conducting the proper experiment two preliminary experiments took place. The point of these experiments was to check if the methodology of the research is appropriate and to correct any, previously unnoticed, flaws in its design. One involved Elodea canadensis Michx. (Canadian waterweed) and the other Glycine max (soybean). The first one has not yielded any statistically relevant data whereas the second one provided data concerning the influence of electric current on the mass of soybeans only. It involved 4 trials (0V, 3V, 6V, 9V) and 8 seeds per each. These experiments did not involve enough samples and trials. Also during the second one mould developed on the seeds due to high humidity of the hydrogel that led (later on) to an improvement of growing medium (transition from pure hydrogel into mixture with soil). The results of the second experiment are discussed later.

4. Variables

• Independent: voltage of applied current (0V, 3V, 6V, 9V, 12V). The voltages in the experiment varied in the following way: for 3V from 2,76-3.03V; for 6V from 5.74-5.87V, for 9V from 8.64-9.07V, for 12V from 11.24-11.73V. The voltages were measured before each electrification. The assumed instead of mean value of voltages is kept throughout this essay to avoid confusion.
• Dependent: germination time assessed by time taken for seeds to germinate (emergence of hypocotyl), post-germination growth assessed by the length of stems and roots of Raphanus sativus L. and stem to root length ratio.

Table 1: Table of controlled variables in the experiment

5. Hypothesis

According to Blackman all plants have their minimum, optimum and maximum levels of voltage which can be applied⁶. To observe outcomes mentioned in the section 2.2 the conditions for all trials should be the same. Then the decisive factor will be the level of voltage applied. One of the levels may be the optimum for radish (Raphanus sativus L.), which should achieve faster growth in such environmental conditions. When the voltage level is sufficient, the growth of the seedlings should be the fastest whereas when larger or lower voltage is applied the growth should decelerate. The expected graph of length of seedlings against voltage should be close to a parabola.

6. Methodology

6.1 Equipment

• 20 plastic cups, volume of 0.5 litres
• 2.5 litres of tap water
• tablespoon
• bowl
• 150 g of hydrogel
• 7.5 litres of garden soil
• 40 radish seeds
• electric cells providing voltage of 3V, 6V, 9V and 12V (separately or in combination^[1])
• sockets with wires for electric cells
• voltmeter [V± 0.01V]
• ruler [cm ± 0.1 cm]
• ammeter [mA±0.01mA]
• electric balance [g±1g].

^[1]Elements in circuits may be connected to one another in two ways: in series or in parallel. The first way should be used as then the voltages of individual electric cells add up¹⁶.

6.2 Procedure

1. Pour 2.5 litres of tap water into the bowl and mix it with 150g of hydrogel.
2. Mix obtained gel with 7.5 litres of garden soil.
3. Distribute the mixture evenly between 20 cups, 0.5 litre per cup.
4. Put 2 seeds of Raphanus sativus L. into each cup, 1 cm apart and 1 cm deep in the soil, overall 8 seeds per voltage level (0V, 3V, 6V, 9V, 12V) applied.
5. Measure the voltage of the batteries using a voltmeter.
6. Water all seeds immediately before electrification, 50 ml per cup.
7. Align wires protruding from sockets in parallel to the seeds and start the stopwatch. Electrify for 5 minutes.
8. Repeat 7th step 15 times changing voltage (3V, 6V, 9V, 12V), omit cups with control group (0V).
9. Repeat steps 5-8 for 11 days.
10. After this time measure the seedlings’ length using a ruler, roots and stems separately.
11. Using Excel (or other spreadsheet) calculate standard deviation (SD), relative standard deviation (RSD) and R² (Pearson correlation coefficient) values, plot graphs of mean length of stems, roots and whole seedlings against voltage applied. Also calculate stem length to root length ratio.
12. Apply statistical tests ANOVA and post-hoc Tukey e.g. using online calculators like: socscistatistics.com, statpages.info.

The hydrogel was used as it not only enhances the growth of plants but also increases the electrical conductivity of the soil³⁵. This enables for creation of a roughly uniform electric field across the seeds.
Time of electrification was established based on the research by Kinney who electrified seeds for a maximum of 5 minutes. In his research it was found that even such a short period of electrification led to significant change in growth²⁰.
Safety issue: the electric current may cause harm if its intensity is larger than 100 mA¹⁷, in this experiment the maximum intensity was around 30mA hence anybody’s health was not endangered.

7. Results

The germination time for every seed was the same as each sample had germinated within 1 day from planting. After that all seeds were only electrified once so it is improbable that electric current had any effect on their germination. Therefore, this analysis will be focused on the post-germination growth.
Table 2: The relationship between the mean length [cm] of Raphanus sativus L. seedlings and different levels of voltage applied (0V, 3V, 6V, 9V, 12V), the length of stem to length of root ratio values and percentage change in length of roots. SD, RSD and mean values are presented.

Standard deviation was calculated using the following formula: . Where ‘x’ is the measured parameter of a radish e.g. length of root and is its mean value, ‘n’ signifies the sample size.

8. Statistical analysis

1. ANOVA one-way test – should be used when there is only one dependent variable and one independent variable²² and it indicates whether in the set of recorded samples of data significant differences between any two pairs of samples have occurred³¹. In this experiment the dependent variables are: the length of stem, the length of root and the total length of radish. The independent variable is the voltage applied.
2. Tukey post-hoc test – follows up the ANOVA as it localises the pair between which the differences occurred³¹.
3. SD and RSD – standard deviation indicates distance of data points from the mean¹³, whereas relative standard deviation shows how data points vary around the mean with comparison to it¹.
4. p value – it indicates the strength of the evidence. If the p-value is less than 0.05 then the evidence is strong enough to confirm the hypothesis of the experiment. If the p-value is greater than 0.05 the evidence is not sufficiently strong.

The results calculated on page Socscistatistics²⁹ for ANOVA test are as follows:

• There are no significant differences between samples’ total length and stems length. The ANOVA test yielded results = 0.77 and = 0.19 respectively (higher than 0.05).
• The result concerning root length is = 0.049 (lower than 0.05) which corresponds to a 95% confidence interval. That means that in 95% of samples their root length lied within the mean value for the entire trial. Hence the values concerning the root length are less likely to be produced by chance. This allows for the further statistical investigation of these results.

Hence the post-hoc Tukey test was conducted only for the length of roots of Raphanus sativus L. and yielded the following results:
Table 3:Results of post-hoc Tukey test. Legend: group 1 -0V, group 2-3V, group 3-6V, group 4-9V, group 5-12V. Calculated on a webpage Socscistatistic.com ²⁹.

As can be seen there is a statistically significant difference between the first and second trials with respect to root length.

9. Data analysis

It is worth mentioning that application of correlation coefficients (R or R²) would be an inaccurate measure of correlation as they apply only to linear relationships, which is not the case in this analysis. The R² coefficient takes values from 0 to 1, the closer it is to 1 the closer data fits a linear trend. The R coefficient is a number between -1 and 1 and analogically to R² it indicates if the trend is linear (values close to either -1 or 1). Moreover, its sign indicates whether the relationship between the two variables is increasing (plus sign) or decreasing (minus sign).
The statistical test applied is the one-way ANOVA test as it examines the statistical significance of the differences between the samples. It is independent of the relationship between the samples and the factor investigated.

Figure 5: Graph of dependence between mean total length of seedlings [cm] of Raphanus sativus L. samples and voltage applied [V]. Error bars source: standard deviation. R²-Pearson Correlation Coefficient. Created using Excel.
Fig.5 presents that there is no quadratic correlation between the total length of seedlings and voltage applied (represented by orange function). Error bars which overlap for each data point indicate that the trend line can as well be a straight line parallel to x-axis (represented by green function, low R² value (0.0176) indicates no correlation). Therefore it may be assumed that voltage applied did not affect the total length of seedlings. The p-value of ANOVA test is greater than 0.05 (p=0.77) (section 8) hence the differences between samples are insignificant.
The next figure (Fig.6) presents data concerning the relationship between mean stem length and voltage applied.

Figure 6: Graph of dependence of mean length of stems of Raphanus sativus L. [cm] on voltage applied [V]. Error bars source: standard deviation. R²-Pearson Correlation Coefficient. Created using Excel.
The overlapping error bars allow for repeated assumption of lack of correlation due to almost constant value of length of stems linearly presented by red function, low R² (0.011) hence no correlation. The explanation of these results may be rooted in the action of auxin. Under undisturbed environmental conditions the presence of auxin causes cells to excrete protons into the apoplast at an enhanced rate. This results in lowered pH which activates wall-loosening processes²⁶. This process allows for elongation of the stem in the plant. If protons were affected by the electric field then the growth of the stem would also be. Lack of electric field across the stem-only across the roots (explanation in the discussion section) probably led to undisturbed movement of H+. This also plausibly affected the overall length of the whole seedling distorting the trend depicted in Fig.5. Due to this fact any measures involving the length of stem are not an accurate description of the relationship of the plant’s growth and the voltage applied. This discards the stem length to root length ratios presented in Tab. 2. Moreover, the results are not significant statistically as the p-value from ANOVA test is greater than 0.05 (p=0.19) (section 8).
The following graph (Fig. 7) represents the correlation between the mean root length and value of voltage applied.

Figure 7: Graph of the relationship between length of roots [cm] of Raphanus sativus L and the voltage applied [V]. Source of error bars: standard deviation. R2-Pearson Correlation Coefficient.
The graph first increases, reaches highest recorded value for the 3V trial and then decreases. This shape is a parabola, however, due to large values of standard deviation the shape of the graph cannot be stated with certainty as due to overlap of error bars, the line from 3V onwards could be a straight line parallel to the x-axis. If the trend is indeed parabolic its peak value should occur for voltage=6.65V as it was calculated from the formula for the blue trend line (equation P in Fig. 7, generated by Excel). However, it is also evident from the graph that the maximum value was measured for the 3V trial as from this point the successive data points have decreasing values. The only valid observation may be made based on the difference between 0 and 3V trials as the difference between them is statistically significant (Tab.3). The greatest increase (40%) in the length of roots occurs between 0 and 3V (data in Tab.2). Also maximum root length of Raphanus sativus L. may occur anywhere between 0V and 3V as there is observable increase in mean root length.
ANOVA test showed that there occurs a statistically significant difference between at least two trials as p-value is lower than 0.05 (p=0.049) (section 8)²⁹. However, the post-hoc Tukey test revealed that only between 0V and 3V trials this difference occurs (p=0.0280)(Tab.3). Because of this, the trend cannot be linear (black function in Fig.7). From the graph in Fig.7 it is evident that the greatest length of root is observed for the 3V trial. It may reflect the increased uptake of minerals like K+ and Ca++ when a current is applied. This may be the effect of electrostimulation of active ion pumps⁵. The stimulation also occurs when the membrane voltage exceeds critical value, then the changes in the membrane occur causing pore formation that facilitates transport³⁷. The results in Fig.7 may be compared to the results of the preliminary experiment with use of soybeans. Although the relationship between mass and voltage applied was investigated it depicts similar graph shape (Fig.8).

Figure 8: Graph of the dependence of mass [g] of soy seedlings Glycine max L. on the voltage applied [V]. Error bars source: standard deviation. R2-Pearson Correlation Coefficient.
Here the maximum mass of seedling should be obtained at voltage=4.1V according to the formula (equation P, Fig.8) but among the data collected the largest mass corresponds to the seeds treated with voltage=3V. The effect on mass may come from water accumulation in cells due to electropermeabilization⁽²⁾ of the cellular membrane as it facilitates diffusion of polar molecules³⁴ and accumulation of sugars and amino acids¹². A similarity can be observed between the graphs in Fig.8 and Fig.7. However, ANOVA test for data in Fig.8 indicated that the results are not statistically significant so the similarity is most probably due to chance.

10. Discussion

The research does not entirely support the hypothesis that there is a relationship between length of roots of Raphanus sativus L. and the voltage applied. There is 95% confidence that the data for 2 different trials are statistically relevant by the post-hoc Tukey test (only concerning roots length). The optimum potential difference for growth of Raphanus sativus L. was found to be 3V, however, it may lie somewhere in the interval 0-3V. Although, due to high RSD of the 6V trial (28%) and lack of significant difference between the 3V and 6V trials the optimum level cannot be determined with high certainty. This research may provide a starting point for further studies on the influence of electric current on the growth of radish and possibly other plants from the Raphanus genus. It has also tested the previously used methodology, provided improvements to it, and some further suggestions are presented in the few next paragraphs. Nonetheless, this study has not provided any justification for any of the explanations proposed in the data analysis section. The hypotheses concerning accumulation of amino acids, and polar molecules^{34, 12} as well as those concerning the activity of proton pumps in the roots should be subjected to further research on the cellular level in order to confirm or reject them as the mechanisms by which electric current acts on plants ^{5, 37}.
Not a whole plant was subject to the electric field resulting in no effect of applied current on the stem of the Raphanus sativus L. Only roots were electrified as the current was flowing exclusively through the growing medium not through the stem. A possible solution would be to use two uniformly charged plates, one placed under the pot and the other fixed above it. The potential difference between the plates may be calculated from the formula where E is the electric field strength [NC-1], V potential difference [V] and d separation [m] of the plates¹⁶. For example, for a voltage of 3V, the electric field strength was 150 Vm-1 the same strength can be obtained in plate arrangement by having a potential difference of 12V between the parallel plates.
Seeds were planted too close to the surface ( ~1cm) of the soil resulting in weak root anchorage and immediate possibility of observing emerging shoots, disabling any determination of germination speed. This discarded electric current as the factor influencing germination as stems in all trials emerged in a short time. Planting the seeds deeper (~ 2cm) may fix the problem.
⁽²⁾When a large potential difference is established across the membrane it becomes more fluid. This in turn allows for insertion of additional proteins. This is called electropermeabilization and its effect lasts even a few minutes after the current was applied³⁴.
The Method of measuring the length of the roots was flawed. The subject of measurements was only the primary root without the lateral ones (roots were tangled so to avoid breaking them, only overall length was measured). This could have distorted the conclusion concerning the length of the roots. Placing the roots in water in order for them to spread and then taking a photo would help improve accurate measurement. In the photograph, it would be possible to measure total root length using a ruler and by calculating scale.
The voltage applied to the samples varied as the experiment progressed due to factors explained later in this section. As a result the electric fields acting on the roots did not have a constant strength. The following factors might have contributed to the variation of the voltage applied. The desired voltages were obtained by connecting the electric cells in series. This accounts for loss of energy on the connections between them. The arrangements were not fixed so that voltages varied between electrifications. Another cause of varying voltage is the voltage drop in an electric cell at the beginning of their action and the other is increasing resistance in the wires. The latter was the effect of the action of hydrogel because the wires tarnished when placed in it. Presence of the soil alleviated this effect but it was still present. The first two problems may be bypassed by using the direct current power supply instead of electric cells so that voltage is kept constant. Exchange of copper wires for carbon electrodes should also eliminate the possibility of increasing resistance.
The roots may have got injured during the picking of radishes from the soil. They were taken out in water together with the soil to avoid breaking but during pulling the fragile parts of the roots might have broken. If the seedlings were left for a longer time (e.g. 22 days) they possibly would develop edible roots which are harder to break so there would be a lower possibility of distorted length measurement. It would also allow for mass measurements.
Based on this evaluation the following extensions to the experiment are proposed.
There should be more samples per one trial e.g. 20 seeds per one potential difference to eliminate anomalies and random variation in seeds tissues.
Conducting an experiment with greater number of potential differences between 0V and 3V to search for a voltage value for which the length is maximum. The more voltages are investigated the more accurate computer models can be created as the current ones are flawed.
Carrying out the experiment with use of charged plates as mentioned in the second paragraph of this section.
As mentioned at the beginning of this section, the investigation of the optimum potential differences for other species e.g. Raphanus caudatus L. and Raphanus raphanistrum L. Could be carried out and one could look for similarities in the trend within the genus Raphanus.
The investigation of electrotropism i.e. the direction of growth of the plant root placed in the electric field based on the paper by Wawrecki and Zagórska-Marek³⁶.

11. Conclusion

The hypothesis of the research was that there will be an optimal voltage for which the radish seedlings will achieve the fastest growth. If the voltage was too low or too high the seedlings’ growth was to decelerate. Because the length of root and shoot of a plant were used as measures of growth the research is inconclusive. For the entire seedlings the differences between trials are not statistically significant, nor are they for the shoot lengths. However, for root lengths there exists a statistically significant difference in length between 0V and 3V trials. This difference corresponds to a 40% increase in root length between these two trials. It is consistent with findings of Black et al. who has observed 5-35% increase in the length of the whole plant (unnamed in paper)⁵. Also, a celery plant (Apium gravaeolens) was found to grow higher when treated with current of voltage 7.5V²⁷. Nonetheless, the research carried out does not allow for a straightforward rejection or acceptance of the entire hypothesis as it requires further experiments. A similar trend was found in a preliminary experiment with use of Glycine max L., where mass of seedlings was largest in 3V trials. It would be similar to a finding that tomato plant fruit yield was 27% greater if cultivated in ionised air, which was achieved by passing electric current through the air using electrodes⁴⁰. However, the trials with soybeans were also not statistically significant as indicated by the ANOVA test.

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About the Author

Tomasz Zuber is a 19 year old Polish student who has graduated from the IB Diploma Programme. He is interested in biology, especially plant biology and evolution and in physics. He intends to pursue both these disciplines at university.