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

Summary

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.

Abstract

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 <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.

Introduction

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 force^3,4. 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 'Komatsuna' 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 growth^6-8, 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.

Protocol

1. Initial Settings

1. Agricultural Plant Species
   1. 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 utilized^5,7.
2. Plant Cultures
   1. 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.
   2. 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 °C, with humidity ranging 65-75% (please check section 3.1.2).
   3. For number of seeds, culture 10 ± 1 seeds of Garden Balsam, 50 ±10 seeds of Mizuna, 330 ± 20 seeds of Komatsuna, and 380 ± 20 seeds of Mescluns. Use identical conditions measured as 18-25 °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.

2. Culture of Four Agricultural Plants

1. Experimental Procedure
   1. Follow section 1.2.3) for species of plant and culture conditions in control and magnet applied group.
   2. Apply three magnets of 1,750 ± 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.
   3. 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.

3. Tubulin Staining of Garden Balsam  

1. Magnet Applications with Regulated Light Condition
   1.  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 °C for 48 hr in dark environments. Eventually, this condition was not used in this experiment due to high variations in growth length.
2. Plant Staining
   1. 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.
   2. 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.
   3. For double immunostaining, incubate the sample with primary antibody, anti-alpha tubulin (1:1,000), O/N at 4 °C.
   4. 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 °C.
   5. 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 (λ = 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.

4. Data Collection Methods

1. Time-lapse Creation of Four Agricultural Plant Growth
   1. 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.
   2. Collect 700-900 pictures for 7-10 days. Connect the camera with electric wires since battery could be depleted.
   3. 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.
   4. 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.
   5. Perform t-test and box plot for statistical analysis^11,12.
      NOTE: Groups of five-number summaries were used to calculate the lower limit (L) value as Q1 - [1.5 × (Q3 - Q1)] and the higher limit (H) value as Q3 + [1.5 × (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's t-test were used to analyze the differences in the heights of seedlings¹².

Results

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).

Discussion

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-ter...

Materials

Name	Company	Catalog Number	Comments
Static magnets	JIM	N/A	2000Gauss
2% horse serum/1% bovine serum albumin/0.1% Triton X-100	Sigma-Aldrich	Merged with 55514	Blocking buffer
Primary antibody	Santa Cruz Biotechnology	sc-8035	a-Tubulin
Secondary antibody	Santa Cruz Biotechnology	sc-2010	FITC-conjugated anti-mouse IgG
time lapse photographic techniques	Manually controlled	N/A	ISO value 400 & aperture F 3.2
Sony Vegas Pro 13.0	Sony	N/A	N/A