Name: enhancement of the initial growth rate of agricultural plants by using static magnetic fields
Uploaded: 2016-07-08T13:00:00.000+00:00
Duration: 5 min 17 s
Description: Watch this Scientific Journal Video about Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields at JoVE.com

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-ter...

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><tbody><tr><td>Static magnets</td><td>JIM</td><td></td><td>2000Gauss</td></tr><tr><td>2% horse serum/1% bovine serum albumin/0.1% Triton X-100</td><td>Sigma-Aldrich</td><td>Merged with 55514</td><td>Blocking buffer</td></tr><tr><td>Primary antibody</td><td>Santa Cruz Biotechnology</td><td>sc-8035</td><td>a-Tubulin</td></tr><tr><td>Secondary antibody</td><td>Santa Cruz Biotechnology</td><td>sc-2010</td><td>FITC-conjugated anti-mouse IgG</td></tr><tr><td>time lapse photographic techniques</td><td>Manually controlled</td><td></td><td>ISO value 400 &amp;amp; aperture F 3.2</td></tr><tr><td>Sony Vegas Pro 13.0</td><td>Sony</td><td></td><td></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).
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Watch this Scientific Journal Video about Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields at JoVE.com

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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13.2K 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

Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) and the moment of the radiation of Angiosperms. This led to the hypothesis that alterations in GMF polarity may play a role in plant evolution. Here, we describe a method to test this hypothesis by exposing Arabidopsis thaliana to artificially reversed GMF conditions. We used a three-axis magnetometer and the collected data were used to calculate the magnitude of the GMF. Three DC power supplies were connected to three Helmholtz coil pairs and were controlled by a computer to alter the GMF conditions. Plants grown in Petri plates were exposed to both normal and reversed GMF conditions. Sham exposure experiments were also performed. Exposed plants were photographed during the experiment and images were analyzed to calculate root length and leaf areas. Arabidopsis total RNA was extracted and Quantitative Real Time-PCR (qPCR) analyses were performed on gene expression of CRUCIFERIN 3 (CRU3), copper transport protein1 (COTP1), Redox Responsive Transcription Factor1 (RRTF1), Fe Superoxide Dismutase 1, (FSD1), Catalase3 (CAT3), Thylakoidal Ascorbate Peroxidase (TAPX), a cytosolic Ascorbate Peroxidase1 (APX1), and NADPH/respiratory burst oxidase protein D (RbohD). Four different reference genes were analysed to normalize the results of the qPCR. The best of the four genes was selected and the most stable gene for normalization was used. Our data show for the first time that reversing the GMF polarity using triaxial coils has significant effects on plant growth and gene expression. This supports the hypothesis that GMF reversal contributes to inducing changes in plant development that might justify a higher selective pressure, eventually leading to plant evolution.

Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) ...

Developmental Biology

Robotic Sensing and Stimuli Provision for Guided Plant Growth

Robot systems are actively researched for manipulation of natural plants, typically restricted to agricultural automation activities such as harvest, irrigation, and mechanical weed control. Extending this research, we introduce here a novel methodology to manipulate the directional growth of plants via their natural mechanisms for signaling and hormone distribution. An effective methodology of robotic stimuli provision can open up possibilities for new experimentation with later developmental phases in plants, or for new biotechnology applications such as shaping plants for green walls. Interaction with plants presents several robotic challenges, including short-range sensing of small and variable plant organs, and the controlled actuation of plant responses that are impacted by the environment in addition to the provided stimuli. In order to steer plant growth, we develop a group of immobile robots with sensors to detect the proximity of growing tips, and with diodes to provide light stimuli that actuate phototropism. The robots are tested with the climbing common bean, Phaseolus vulgaris, in experiments having durations up to five weeks in a controlled environment. With robots sequentially emitting blue light-peak emission at wavelength 465 nm-plant growth is successfully steered through successive binary decisions along mechanical supports to reach target positions. Growth patterns are tested in a setup up to 180 cm in height, with plant stems grown up to roughly 250 cm in cumulative length over a period of approximately seven weeks. The robots coordinate themselves and operate fully autonomously. They detect approaching plant tips by infrared proximity sensors and communicate via radio to switch between blue light stimuli and dormant status, as required. Overall, the obtained results support the effectiveness of combining robot and plant experiment methodologies, for the study of potentially complex interactions between natural and engineered autonomous systems.

Distributed robot nodes provide sequences of blue light stimuli to steer the growth trajectories of climbing plants. By activating natural phototropism, the robots guide the plants through binary left-right decisions, growing them into predefined patterns that by contrast are not possible when the robots are dormant.

Robot systems are actively researched for manipulation of natural plants, typically restricted to agricultural automation activities such as harvest, ...

Engineering

Control and Disposal of Invasive Japanese Knotweed Reynoutria japonica Houtt. Using Microwave Treatment

The study aims to assess the effectiveness of microwave treatment (MWT) at a frequency of 2.45 GHz and a power of 800 W to control Japanese knotweed (Reynoutria japonica Houtt.) using a self-propelled device that was built in the in-house facility. The MWT was applied in the field population of knotweed in July 2022. First, plants were mechanically moved from the area of 1 m2, and next, the cut shoots around 4 cm high were microwave-treated for 25 min, 20 min, and 15 min. The control treatments were: 1) only cut plants and 2) rhizomes dug out to 30 cm deep. The effectiveness of the microwave treatments was observed for the next 11 months by counting the number of newly grown shoots. The results showed that a 25 min MWT was 100% effective in Japanese knotweed loss of vitality, while a 15 min MWT microwave treatment stimulated plant growth by around 50%, compared to controls. Rhizomes were dug out in a separate in vitro experiment for laboratory testing. The rhizomes were categorized by thickness and subjected to a 60 s MWT using a commercial microwave, after which their temperature and vitality were assessed. The temperature of rhizomes following MWT depended on their thickness. Those rhizomes that warmed to temperatures above 42 &#176;C were effectively destroyed. Summing up, the time plants are exposed to microwaves plays a major role in the effectiveness of this method. The longer the exposure to MWT, the better control. The thinner the rhizomes, the more effective the in vitro MWT rhizomes disposal.

This protocol provides a method for microwave control of Japanese knotweed in the field and disposal of dug-up rhizomes in laboratory conditions. The advantages and disadvantages of both methods are discussed. Future research directions are also suggested to optimize the use of microwaves for controlling Japanese knotweed.

The study aims to assess the effectiveness of microwave treatment (MWT) at a frequency of 2.45 GHz and a power of 800 W to control Japanese knotweed ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Bioengineering

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Neuroscience

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Biology

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Stimulation of Patterned Neuronal Cultures by a Magnetic Field

Source: Stern, S., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54357">External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures.</a> J. Vis. Exp. (2017)
This video demonstrates a method for stimulating neuronal cultures grown in a patterned circular ring on a coverslip, using a time-varying magnetic field.

1. Preparation of coverslips for 1D cultures.

	Clean glass coverslips by immersion in a base piranha solution consisting of 75 mL DDW, 25 mL 25% ammonia ...

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

Summary

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3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

Stimulation of Patterned Neuronal Cultures by a Magnetic Field