This study received supported from the National Research Foundation of Korea (NRF) (2011-0012728). A poster presenting this study was awarded the Best Poster Award by the Korean Society of Applied Biological Sciences (KSABC).

1. Initial Settings
<ol>
	<li>Agricultural Plant Species
	<ol>
		<li>Use Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mescluns (Lepidium sativum) seeds. 
		NOTE: Impatiens balsamina (Garden Balsam or Rose Balsam) is a species native to India; a few members are also located in Myanmar. Komatsuna (Brassica rapa var. perviridis or komatsuna) is a variant of the same species as the common turnip. Garden cress (Lepidium sativum) is a type of herb that is taxonomically related to watercress and mustard. They have similar flavors and scent, for which they are commercially utilized5,7.</li>
	</ol>
	</li>
	<li>Plant Cultures
	<ol>
		<li>Culture Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mescluns (Lepidium sativum) seeds in a 100 mm diameter (100 pi) Petri dish. Ensure that one plate contains only one type of species.</li>
		<li>For culture conditions, place the seeds on a cellulose towel. Immerse the towel and seeds in triple distilled water. Measure and confirm that the indoor lab RT is 18-25 &#176;C, with humidity ranging 65-75% (please check section 3.1.2).</li>
		<li>For number of seeds, culture 10 &#177; 1 seeds of Garden Balsam, 50 &#177;10 seeds of Mizuna, 330 &#177; 20 seeds of Komatsuna, and 380 &#177; 20 seeds of Mescluns. Use identical conditions measured as 18-25 &#176;C, with humidity ranging 65-75% (please check section 2.1.1). 
		NOTE: All experiments were performed on indoor conditions with the regulated humidity and temperature range in lab. The humidity and temperature was not static, but provided the identical conditions for magnet treated group and control.</li>
	</ol>
	</li>
</ol>
2. Culture of Four Agricultural Plants
<ol>
	<li>Experimental Procedure
	<ol>
		<li>Follow section 1.2.3) for species of plant and culture conditions in control and magnet applied group.</li>
		<li>Apply three magnets of 1,750 &#177; 350 Gauss (10,000 Gauss = 1 Tesla) at the bottom of the 100 pi dishes for Garden Balsam. During the application, ensure that the three magnets are not in direct contact with the seeds, and are separated by the plastic bottom of the petri dish. The direct distance between seeds and magnets should be 2-4 mm. Apply magnets for 168 hr (7 days) for four agricultural plants.</li>
		<li>Following all steps identically in 2.1.2), apply two magnets, one (facing N side upward) on the top and other magnet (facing S side upward) on the bottom of Garden Balsam culture plate. 
		NOTE: The poles are applied differently in Garden Balsam. However, the pole orientation is not considered as a crucial factor in this study for growth alterations, as all environments are identical except for the direction of the magnetic flux. The purpose of N and S pole application for Garden Balsam was to see its practical ability in using it in fields, where pole orientation could be hard to manage.</li>
	</ol>
	</li>
</ol>
3. Tubulin Staining of Garden Balsam &#160;
<ol>
	<li>Magnet Applications with Regulated Light Condition
	<ol>
		<li>&#160;Place two magnets (N pole facing up) bottom of the 100 mm plate for 48 hr, using conditions in step 1.2.2. 
		NOTE: For modification of light, the culture dishes were placed on a plastic shelf in the incubator. Incubator was used for interception of light and maintaining temperature at 25 &#176;C for 48 hr in dark environments. Eventually, this condition was not used in this experiment due to high variations in growth length.</li>
	</ol>
	</li>
	<li>Plant Staining
	<ol>
		<li>Fix the entire impatiens SPP double flower plant, (including stem and roots) grown on identical conditions with step 3.1.2) in 4% paraformaldehyde and 0.1 M phosphate buffer (pH 7.4) for 15 min.</li>
		<li>Remove the impatiens sample and immerse for 2 hr in blocking buffer (2% horse serum/1% bovine serum albumin/0.1% Triton X-100 in PBS, pH 7.5). Wash the impatiens sample by immersing with PBS for 15 min.</li>
		<li>For double immunostaining, incubate the sample with primary antibody, anti-alpha tubulin (1:1,000), O/N at 4 &#176;C.</li>
		<li>Remove the sample and immerse the samples once with PBS for 10 min to wash. Use FITC-conjugated anti-mouse IgG (1:400) as the secondary antibody and incubate for 2 hr at 25 &#176;C.</li>
		<li>Immerse the sample in PBS and cover slip the whole sample in bottom of 24 well plate. Obtain images using a conventional fluorescence microscope to observe tubulin orientation (&#955; = 550 nm, magnified to 100X, 200X and 400X). 
		NOTE: In this case, the magnet treated group (n = 10) and controls (n = 11) were confirmed for Garden Balsam (Impatiens balsamina) only, grown in non-dark conditions.</li>
	</ol>
	</li>
</ol>
4. Data Collection Methods
<ol>
	<li>Time-lapse Creation of Four Agricultural Plant Growth
	<ol>
		<li>Photograph the plant at 10 min intervals, by setting shutter to auto (this can be done in any digital camera). Set the aperture to F 3.2 and the ISO value to 400.</li>
		<li>Collect 700-900 pictures for 7-10 days. Connect the camera with electric wires since battery could be depleted.</li>
		<li>Drag the pictures by clicking and dropping each picture chronologically under the streaming line with moviemaking software (see materials and equipment table). Put it on a streaming line in equal durations of 0.045-0.05 sec for each into a film total of 30-40 sec. Check so that there are no dark gaps with selecting the each picture in a chronological order.</li>
		<li>After step 4.1.3, click play button in software to ensure the compiled-movie into a 30-40 sec time-lapse video slide and click render and save to .mpeg or .avi format. For size markers, use Canadian Quarter, an American Penny, and a centimeter ruler on the side of the photo.</li>
		<li>Perform t-test and box plot for statistical analysis11,12. 
		NOTE: Groups of five-number summaries were used to calculate the lower limit (L) value as Q1 - [1.5 &#215; (Q3 - Q1)] and the higher limit (H) value as Q3 + [1.5 &#215; (Q3 - Q1)]. This approach was incorporated into step 1.2.2 for length data collection11. The L and H values show the 99% area of the T-distribution plots, which means that the data points observed outside this range can be considered outliers. Box plots and Student&#39;s t-test were used to analyze the differences in the heights of seedlings12.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Martin, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+measurement+of+pollen+tube+growth+inhibition.">In vitro measurement of pollen tube growth inhibition.</a> Plant Physiol. 49, 924-925 (1972).</li><li>Pfahler, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+germination+characteristics+of+maize+pollen+to+detect+biological+activity+of+environmental+pollutants.">In vitro germination characteristics of maize pollen to detect biological activity of environmental pollutants.</a> Environ Health Perspect. 37, 125-132 (1981).</li><li>Yao, Z., Tan, X., Du, H., Luo, B., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-current+microwave+ion+source+with+permanent+magnet+and+its+beam+emittance+measurement.">A high-current microwave ion source with permanent magnet and its beam emittance measurement.</a> Rev Sci Instrum. 79, 073304 (2008).</li><li>Hendrickson, C. L., Drader, J. J., Laude, D. A., Guan, S., Marshall, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fourier+transform+ion+cyclotron+resonance+mass+spectrometry+in+a+20+T+resistive+magnet.">Fourier transform ion cyclotron resonance mass spectrometry in a 20 T resistive magnet.</a> Rapid Commun Mass Spectrom. 10, 1829-1832 (1996).</li><li>Namba, K., Sasao, A., Shibusawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EFFECT+OF+MAGNETIC+FIELD+ON+GERMINATION+AND+PLANT+GROWTH.">EFFECT OF MAGNETIC FIELD ON GERMINATION AND PLANT GROWTH.</a> Acta Hort. 399, 143-148 (1995).</li><li>Hirota, N., Nakagawa, J., Kitazawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+magnetic+field+on+the+germination+of+plants.">Effects of a magnetic field on the germination of plants.</a> Journals of Applied Physics. 85, 5717-5719 (1999).</li><li>Penuelas, J., Llusia, J., Martinez, B., Fontcuberta, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diamagnetic+Susceptibility+and+Root+Growth+Responses+to+Magnetic+Fields+in+Lens+culinaris,+Glycine+soja,+and+Triticum+aestivum.">Diamagnetic Susceptibility and Root Growth Responses to Magnetic Fields in Lens culinaris, Glycine soja, and Triticum aestivum.</a> Electromagnetic Biology and Medicine. 23, 97-112 (2004).</li><li>Carbonell, M. V., Martinez, E., Amaya, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+germination+in+rice+(Oryza+Sativa+L.)+by+a+static+magnetic+field.">Stimulation of germination in rice (Oryza Sativa L.) by a static magnetic field.</a> Electro- and Magnetobiology. 19, 121-128 (2000).</li><li>Oakley, R. V., Wang, Y. S., Ramakrishna, W., Harding, S. A., Tsai, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expansion+and+expression+of+alpha-+and+beta-tubulin+gene+families+in+Populus.">Differential expansion and expression of alpha- and beta-tubulin gene families in Populus.</a> Plant Physiol. 145, 961-973 (2007).</li><li>Hoson, T., Matsumoto, S., Soga, K., Wakabayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+microtubules+are+responsible+for+gravity+resistance+in+plants.">Cortical microtubules are responsible for gravity resistance in plants.</a> Plant Signal Behav. 5, 752-754 (2010).</li><li>Kim, S., Im, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Static+magnetic+fields+inhibit+proliferation+and+disperse+subcellular+localization+of+gamma+complex+protein3+in+cultured+C2C12+myoblast+cells.">Static magnetic fields inhibit proliferation and disperse subcellular localization of gamma complex protein3 in cultured C2C12 myoblast cells.</a> Cell Biochem Biophys. 57, 1-8 (2010).</li><li>Benjamini, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opening+the+Box+of+a+Boxplot.">Opening the Box of a Boxplot.</a> The American Statistician. 42, 257-262 (1988).</li></ol>

The Authors have nothing to disclose.

In all conditions, magnets should be applied under the petri dish. This study examined the influence of magnetic fields on growth rate of seeds for several agricultural species, with focus on Garden Balsam as a representative of agricultural plants. For instance, tubulin staining was performed on Garden Balsam to evaluate the molecular-level changes in root and stem skeletal micro-structures suggesting influence of the magnetic field in length proliferation. Both the N and S poles of the magnet were applied in a long-term (7-10 d) follow-up study using Garden Balsam. Three other species, Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mesclun (Lepidium sativum), were treated with N-pole oriented magnets. This was to further verify that the static magnetic field itself, not the poles, was a major factor in initial growth enhancement. Additionally, increasing the number of species provides support for broader applicability of magnet-derived initial growth facilitation in agricultural plants.
Many factors, such as nutrition, humidity, temperature, and light, might affect the rate of plant growth 3. Each of these was held constant across treatments. Nutritional supplement was excluded by only culturing in triple-distilled water. We first controlled for light-experiments were initially performed on Garden Balsam in an incubator under dark conditions. Growth patterns in the dark environment differed from those in the light environment. Therefore, we conducted subsequent experiments under light conditions (using identical amounts of light in all experimental groups). For the tubulin staining, the Garden Balsam was grown under controlled conditions (triple-distilled water, temperature 18-25&#176;C, humidity 65%-75%). Other experiments of the 7-10-d follow-up study had identical &#34;null conditions: no nutrition&#34; conditions to those used with Garden Balsam (triple distilled water, temperature 18-25&#176;C, humidity 65%-75%). With respect to magnet application, we used a strategy wherein we quantitatively increased the number of species and the duration of the magnet application to further investigate whether magnetic fields have a universal growth-facilitating effect on agricultural plants that is not limited to certain species. This idea was investigated using Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis) and Mescluns (Lepidium sativum).
The molecular basis of this phenomenon was partially clarified by the tubulin staining experiments9-11, but further investigation is needed for practical use. Precise magnetic application can be limited in humid environments owing to erosion of the magnet itself. The magnetic fields physically enhance the growth of agricultural plants. However, this does not prove that nutritional content also increases. Further analysis of the chemical contents of the plants should be conducted in order to determine whether the use of magnetic fields has an effect similar to that of a fertilizer. This could also be evaluated in environments in which nutrients are provided, as well as the nutrient-null conditions using distilled water that were employed in the present study. In addition to the quality (type, intensity, etc.) and quantity of applied magnets, cost can be another issue complicating such applications. It can be expensive to apply numerous magnets throughout an entire crop field.
Our findings suggest that static magnetic field application accelerates the germination rate and initial growth rate of several cultivated plant species. These findings demonstrate that a static magnetic field has a significant effect on plant growth, especially the germination 6 and root growth 7 of plants. Previous studies have suggested that maximum germination rates were 20% higher when the frequency of the magnetic field was approximately 10 Hz 5-6. During only 4 d of application of a magnetic field, the stem and root length increased. The group subjected to a magnetic field treatment (n = 10) showed a 1.4-times higher rate of growth than did the control group (n = 11) in a total of 8 d (p &#60; 0.0005). This rate was 20% higher than that found in previous studies that used a pulsed magnetic field 6-9.
Given these findings, gene expression and regulation should also be studied in future experiments for clarification of the potential mechanisms behind the observed responses to magnetic fields10. Our findings suggest that the application of a magnetic field could increase crop growth rate, which could potentially help to address food and poverty issues worldwide. Moreover, the application of a magnetic field might be useful to reduce the use of chemical fertilizers.

Germination is the growth of a plant that results in the formation of the seedling1. Under certain conditions, seed germination begins and the embryonic tissues resume growth. It begins with hydration to the seed in order to activate enzymes for germination. Seeds can be induced to germinate in vitro (in a Petri dish or test tube)1,2.
Static magnetic fields are special forces that cause movements of molecules with ionic charges by way of the Lorentz force3,4. Lorentz force is formed when an ionized or charged object moves under a magnetic field. Every material is formed with atoms which are composed of electrons and protons. When magnetic fields become present, whether it is static or alternating, it affects the movement of charged material. This also applies to plants and water molecules, which affects the intracellular molecule condition. In a previous study, electromagnetic coils were used to generate pulsed magnetic fields, and &#39;Komatsuna&#39; plants were chosen as the subjects5. In the present study, magnet generated static magnetic fields were used to give a similar but different effects as an expansion study of Lorentz force.
The frequency of the magnetic field, rather than its polarity, is a crucial factor for plant germination. Previous studies have suggested that maximum germination rates were 20% higher than control when the frequency of the magnetic field was approximately 10 Hz. When the field was removed in a retrograde manner, the growth rate was impaired5. Static magnetic fields have a considerable effect on the initial growth6-8, primarily on germination6 and root growth7.
In the present study, we used static magnets to examine the possibility of regulating the growth of agricultural plants by using magnetic fields. In particular, we aimed to determine whether certain conditions of magnetic field application could increase the growth rates to higher levels than those mentioned in the literature. Furthermore, if the initial germination of plants can be successfully increased using a magnetic field, the use of chemical fertilizers can be avoided.

<table border="0" cellpadding="0" cellspacing="0" width="646">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Static magnets</td>
			<td style="width:144px;">JIM</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">2000Gauss</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">2% horse serum/1% bovine serum albumin/0.1% Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">Merged with 55514</td>
			<td>Blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-8035</td>
			<td>a-Tubulin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-2010</td>
			<td>FITC-conjugated anti-mouse IgG</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">time lapse photographic techniques</td>
			<td style="width:144px;">Manually controlled</td>
			<td style="width:119px;">N/A</td>
			<td>ISO value 400 &#38; aperture F 3.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sony Vegas Pro 13.0</td>
			<td style="width:144px;">Sony</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">N/A</td>
		</tr>
	</tbody>
</table>

enhancement of the initial growth rate of agricultural plants by using static magnetic fields

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing seeds of Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mescluns (Lepidium sativum). We applied magnets under the culture dish. During the 4 days of application, we observed that the stem and root length increased. The group subjected to magnetic field treatment (n = 10) showed a 1.4 times faster rate of growth compared with the control group (n = 11) in a total of 8 days (p &#60;0.0005). This rate is 20% higher than that reported in previous studies. The tubulin complex lines did not have connecting points, but connecting points occur upon the application of magnets. This shows complete difference from the control, which means abnormal arrangements. However, the exact cause remains unclear. These results of growth enhancement of applying magnets suggest that it is possible to enhance the growth rate, increase productivity, or control the speed of germination of plants by applying static magnetic fields. Also, magnetic fields can cause physiological changes in plant cells and can induce growth. Therefore, stimulation with a magnetic field can have possible effects that are similar to those of chemical fertilizers, which means that the use of fertilizers can be avoided.

Tubulin staining showed dispersed or thinned structures in plants grown in the presence of the magnet compared to the control (Figure 2). Moreover, 7 day time-lapse studies with agricultural plants including Komatsuna (Brassica rapa var. perviridis) and Mescluns (Lepidium sativum) indicated that a magnet derived static magnetic field increases the initial growth of these plants (Figure 3).
These results suggest that the group exposed to a magnetic field had a notable growth change (Figure 1). Plants grown in dark environments did not show any difference, suggesting that only the light existing condition was applicable in a 7 day time-lapse experiment. Three representative agricultural plants were used in this study, but more plants could have been used. Crops and other plants may be investigated using the same protocol. In previous studies, the growth rate increased by 20%, whereas the present results showed a 1.4-fold increase, which is 40%. Thus, application of a magnet with static magnetic field was more effective than the application of alternating magnetic pulses.
Determining the effect of a magnetic field can be complicated because any molecular structure with electric charge may be influenced3,4. The static magnetic field appeared to influence the initial growth rate of garden balsam in a cultured cellulose tower tissue. The value was statistically significant and was approximately 1.4 times the growth rate of the control. Tubulin is essential for maintaining plant structure during cell elongation and growth9.
<img alt="Figure 1" src="/files/ftp_upload/53967/53967fig1.jpg" /> 
Figure 1.&#160;Growth of Garden Balsam. (A) The growth of Garden Balsam treated with a static magnetic field under dark conditions was marginal; however, the plants grew faster when exposed to light (the only representative picture shown). (B) When exposed to light, on day 3, the difference in height was statistically significant (p &#60;0.01, two-sided t-test). (C) Each individual plant&#39;s height was higher until day 7 (**: upper bound of standard error for measurement). Dark conditions did not induce any differences, indicating that the effects of the magnetic field could be associated with hormones. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53967/53967fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53967/53967fig2.jpg" /> 
Figure 2. Tubulin staining of Garden Balsam and the increase in the growth rate of Garden Balsam after the application of a magnetic field. (A) Garden Balsam showed a dispersed distribution of tubulin structure when a magnetic field was applied. This finding indicates that growth-arresting protein structures like tubulin (and possibly actin) are affected by static magnetic fields. (B) The mean growth rate was 1.4-fold higher than that of the control, and the mean height was higher in the group treated with the magnetic field. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53967/53967fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53967/53967fig3.jpg" /> 
Figure 3. A magnetic field facilitated the growth of Mescluns (Lepidium sativum, front) and Komatsuna (Brassica rapa var. perviridis). The seed supplemented petri dish was treated with a magnetic field of 1,750 &#177; 350 Gauss and observed for 7 days with a time-lapse interval of 10 min. The time-lapse video was cut to 15 fragments of 11 hr each. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53967/53967fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1 Frame" src="/files/ftp_upload/53967/53967video1.jpg" /> 
Supplementary Video 1: Growth timelapse of Garden Balsam (Impatiens balsamina). A Garden Balsam (Impatiens balsamina) seed supplemented Petri dish was treated with a magnetic field of 1,750 &#177; 350 Gauss and then observed for 7 days with a time-lapse interval of 10 min. The video was rearranged into a 30 min film. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53967/timelapse1.mp4">Please click here to view this video. (Right-click to download.)</a>
<img alt="Video 2 Frame" src="/files/ftp_upload/53967/53967video2.jpg" /> 
Supplementary Video 2: Comparison of the growth of 3 plants grown in triplicate. Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mesclun (Lepidium sativum) seeds are shown in a 100 mm diameter (100 pi) Petri dish. Under identical conditions to those of Garden Balsam, the three species were evaluated separately, which revealed that the magnet&#39;s effect is broadly observed in agricultural plants. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53967/timelapse2.avi">Please click here to view this video. (Right-click to download.)</a>

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Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields

The goal of this protocol is to demonstrate the acceleration of the initial growth rate of plants by applying static magnetic fields with no external energy.

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing ...

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13.1K Views.  Yonsei University. The goal of this protocol is to demonstrate the acceleration of the initial growth rate of plants by applying static magnetic fields with no external energy.

Video: Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Continuous Instream Monitoring of Nutrients and Sediment in Agricultural Watersheds

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in water resources is a prerequisite for understanding the drivers of the pollutant loads and for making informed water resource management decisions. The commonly used &#34;grab sampling&#34; method provides the concentrations of pollutants at the time of sampling (i.e., a snapshot concentration) and may under- or overpredict the pollutant concentrations and loads. Continuous monitoring of nutrients and sediment has recently received more attention due to advances in computing, sensing technology, and storage devices. This protocol demonstrates the use of sensors, sondes, and instrumentation to continuously monitor in situ nitrate, ammonium, turbidity, pH, conductivity, temperature, and dissolved oxygen (DO) and to calculate the loads from two streams (ditches) in two agricultural watersheds. With the proper calibration, maintenance, and operation of sensors and sondes, good water quality data can be obtained by overcoming challenging conditions such as fouling and debris buildup. The method can also be used in watersheds of various sizes and characterized by agricultural, forested, and/or urban land.

With the advancement of technology and the rise in end-user expectations, the need and use of higher temporal resolution data for pollutant load estimation has increased. This protocol describes a method for continuous in situ water quality monitoring to obtain higher temporal resolution data for informed water resource management decisions.

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in ...

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Assessment of Methane and Nitrous Oxide Fluxes from Paddy Field by Means of Static Closed Chambers Maintaining Plants Within Headspace

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based on the diffusion theory. A known volume of air overlaying a defined soil area is enclosed within a parallelepiped cover (named &#34;chamber&#34;), for a defined period of time. During this enclosure period, gases (methane (CH4) and nitrous oxide (N2O)) move from soil pore air near their microbial source (i.e., methanogens, nitrifiers, denitrifiers) to the chamber headspace, following a natural concentration gradient. Fluxes are then estimated from chamber headspace concentration variations sampled at regular intervals throughout the enclosure and then analyzed with gas chromatography. Among the techniques available for GHG measurement, the static closed chamber method is suitable for plot experiments, as it does not require large homogenously treated soil areas. Furthermore, it is manageable with limited resources and can identify relationships among ecosystem properties, processes, and fluxes, especially when combined with GHG driving force measurements. Nevertheless, with respect to the micrometeorological method, it causes a minimal but still unavoidable soil disturbance, and allows a minor temporal resolution. Several phases are key to the method implementation: i) chamber design and deployment, ii) sample handling and analyses, and iii) flux estimation. Technique implementation success in paddy fields demands adjustments for field flooding during much of the cropping cycle, and for rice plant maintenance within the chamber headspace during measurements. Therefore, the additional elements to be considered with respect to the usual application of non-flooded agricultural soils consist of devices for: i) avoiding any unintended water disturbance that could overestimate fluxes, and ii) including rice plants within the chamber headspace to fully consider gases emitted through aerenchyma transportation.

The overall goal of this protocol is to measure greenhouse gas emissions from paddy fields using the static closed chamber technique. The measurement system needs specific adjustments due to the presence of both a permanent water layer in the field and of the plants within the chamber headspace.

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based ...

Determination of the Settling Rate of Clay/Cyanobacterial Floccules

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction of clay particles with reactive, planktonic cyanobacterial cells to the sedimentary record is under studied. This interaction is a potentially major contributor to shale depositional models. Within a lab setting, the flocculation and sedimentation rates of these materials can be examined and measured in a controlled environment. Here, we detail a protocol for measuring the sedimentation rate of cyanobacterial/clay mixtures. This methodology is demonstrated through the description of two sample experiments: the first uses kaolin (a dehydrated form of kaolinite) and Synechococcus sp. PCC 7002 (a marine coccoid cyanobacteria), and the second uses kaolin and Synechocystis sp. PCC 6803 (a freshwater coccoid cyanobacteria). Cyanobacterial cultures are mixed with varying amounts of clay within a specially designed tank apparatus optimized to allow continuous, real-time video and photographic recording. The sampling procedures are detailed as well as a post-collection protocol for precise measurement of chlorophyll a from which the concentration of cyanobacterial cells remaining in suspension can be determined. Through experimental replication, a profile is constructed that displays sedimentation rate.

The interaction and sedimentation of the clay and bacterial cells within the marine realm, observed in natural environments, can be best investigated in a controlled lab environment. Here, we describe a detailed protocol, which outlines a novel method for measuring the sedimentation rate of clay and cyanobacterial floccules.

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction ...

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less attention has been paid on how environmental factors affect these insects when they pupate within the soil. The tea looper, Ectropis grisescens Warren (Lepidoptera: Geometridae), is a severe pest of tea plants and has caused huge economic losses in South China. The protocols described here aim to investigate, through multiple-choice bioassays, whether mature last-instar E. grisescens larvae can discriminate soil variables such as the substrate type and moisture content, and determine, through no-choice bioassays, the impact of the substrate type and moisture content on pupation behaviors and the emergence success of E. grisescens. The results would enhance the understanding of the pupation ecology of E. grisescens and may bring insights into soil-management tactics for suppressing E. grisescens populations. In addition, these bioassays can be modified to study the influences of various factors on the pupation behaviors and survivorship of soil-pupating pests.

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Here, we present a protocol to investigate the pupation preference of mature larvae of Ectropis grisescens in response to soil factors (e.g., substrate type and moisture content) using choice bioassays. We also present a protocol of no-choice bioassays to determine the factors that affect the pupation behaviors and survivorship of E. grisescens.

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less ...

Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is an accumulating evidence showing that the expressions of certain miRNAs are altered when grafting, an agricultural practice commonly used by farmers to improve crop tolerance to biotic and abiotic stresses. Bottle gourd is an inherently climate-resilient crop compared to many other major cucurbits, including watermelon, rendering it one of the most widely used rootstocks for the latter. The recent advancement of high-throughput sequencing technologies has provided great opportunities to investigate cold-responsive miRNAs and their contributions to heterograft advantages; yet, adequate experimental procedures are a prerequisite for this purpose. Here, we present a detailed protocol for efficiently generating homo- and heterografts between the cold-susceptible watermelon and the cold-tolerant bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis. The presented methods are also useful for other plant-grafting systems, to interrogate miRNA regulations under various environmental stresses, such as heat, drought, and salinity.

Here we present a detailed protocol for efficiently making homo- and heterografts between watermelon and bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis, for the investigation of cold-responsive microRNAs.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is ...