沙漠研究 23-4, 199 -204 (2014 )－APCSEET2013 Refereed Papers－

Journal of Arid Land Studies

Short Time Measurement of R. sativus Root Growthwith Application of A.C. Electric Field
Seiichi SUZUKI *1), Tomoyo HOTTA1), Hideki SUGANUMA1), Shigeru KATO1) and Toshinori KOJIMA1)
Abstract: The effect of a.c. electric field on plant root growth has been investigated for application to the forestation in arid land. In
previous studies, statistic analysis of root growth data has suggested possibility of electric growth enhancement in R. sativus.
However, large deviation by the difference of individual plant and fluctuations in long term culture disturbed the clear demonstration of
the electric effects. To avoid the difficulty of individual differences, magnified time lapse image capture system was constructed forroot growth measurement. This system provides growth measurement within a few minutes, and enables comparison of growth rates
with/without electric field in an experiment of single plant specimen, within a short period of time. Root growth measurement, withspatial resolution of 4 µm, was enabled with this system. Root growth rate was determined in a 5 minutes measurement with lessthan 10% standard deviation. Electric effect on root growth was also measured by this system. The results suggested the growthenhancement by the electric field of 100 - 300 V/m, and inhibition by the electric field of over 500 V/m.
Key Words: Electric field, Root growth, Time lapse image.

1. Introduction
Growing emission of carbon dioxide is one of the mostimportant issue to be addressed by human society. Asidefrom the climate changes by global warming, increasing CO2
concentration is a threat for human health and for the life ofother species, such as seashells or coral. To mitigate theheavy burden of CO2 emission, enhancement of the natural
adsorption by green plant is a good option. In such a purpose,
forestation of arid land is studied in places (Tanouchi et al.,2008). In the case of planting tree seedlings in arid land,
survival rate is largely depending upon the water supply.
Especially, utilization of underground water is crucial in arid
region. Enhancing root growth and leading to the underground water will contribute to plant survival.
Root growth enhancement by application of electric fieldhas been investigated for some plant species (Brayman andMiller, 1986; Suzuki et al., 2002). In our previous study, root
growth enhancement of E. camaldulensis (Eucalyptus tree)
seedling was investigated (Hotta et al., 2012). It showed atendency of root growth enhancement by the a.c. electric fieldof specific intensity. However, clear demonstration was
difficult because individual differences of the plant were sodiverse and fluctuation of growth rate will occur in a long termmeasurement.
In previous studies, a root growthmeasurement session takes some days because root growth rate
* Corresponding Author: seiichi@st.seikei.ac.jp
3-3-1 Kichijoji-Kiamachi, Musashinoshi, Tokyo 180-8633 Japan
1) Dept. Materials and Life Science, Faculty of Science and Technology, Seikei University

is so small. And it also requires many samples to average theindividual difference.
These experimental constrictions
enforce the difficulty in discrimination of slight difference byan experimental parameter. To avoid such problems in plant
culture and to clarify the effect of electric field on root growth,a short time growth measurement system was constructed inthis study. For evaluation of the system, R. sativus (Radish)was used for test specimen. The root growth enhancement bya.c. electric field has been well established for R. sativus so far(Suzuki et al., 2001). Magnified images of a R. sativus root,
growing in water culture vessel, was captured with interval of 1
minute. Root growth rate was analyzed from the difference
between the images. Growth rate measurement, within someminutes, was demonstrated with this system. Effect of a.c.
electric field on the growth of R. sativus root was measured forvarious field intensities. Appropriate time window formeasurement of the growth rate with minimum deviation was
also investigated.

2. Materials and Methods
2.1. Water culture of R. sativus seedling
The R. sativus seedlings were water cultured for opticalobservation of root growth. The inner dimensions of theculture vessel are 100 mm × 120 mm × 50 mm, which is madeof 10 mm thick transparent acrylic plates. Only front side
window was 2 mm thick (Fig. 1). 2 mm thick front
compartment was separated by a perforated back plate from（Received, August 29th, 2013; Accepted, March 3rd, 2014）

Fig. 1. The schematic of the culture vessel for electric field application in solution. Culture vessel is made by 10 mm thick acrylic resin plate. 2
mm gap of front compartment is separated by perforated back plate from bulk medium. Pt electrodes are installed on top and bottom of the front
compartment. Plant seedling was placed in a acrylic pocket on top of the front compartment. The plant root is let to grow through a bottom holeof the pocket into the front compartment.

the bulk medium to guide the root grow straight. A small
pocket was placed on top of the front compartment, having abottom hole for the root to grow through. The bottom of thepocket was covered with a strip of cotton gauze sheet toprevent the seedling from falling down. The culture medium
was Hyponex (HYPONeX Japan Co.), 10,000 times diluted
with deionized water. The conductivity of the Hyponex
solution was kept under 60 µS/cm by exchanging the medium
to suppress the Joule heating. For the test specimen, R.
sativus var. longipinnatus was purchased from Atariya Noen
Co. Cultured plant was illuminated by Bio Fluorescent tube of
NEC with illuminance of about 2400 lux. Room temperatureand humidity were kept 25˚C and 60%, respectively, by air
conditioner. During the growth rate measurement, all thesystem was placed in a closed room and illuminated by LED

Fig. 2. R. sativus seedling in the water culture vessel. A seedling wasplaced in the acryl pocket on top of the culture vessel. The
growing root tip of the seedling can be seen through transparent
acrylic window. The root go down through a hole at the bottomof the pocket and grow along the narrow channel.

light with constant luminance.
For the water culture, 35 seeds of R. sativus were sowed ona folded cotton gauze sheet dipped in deionized water for
germination of the root. After 23 hours, one of 35 seedlings
was picked up to transfer to the culture vessel. It was placedin the pocket of water culture vessel and let grown for 22 hours
before the measurement. For accurate growth measurement,
the front compartment was restricted to 3 mm width by 2 mm
thick plates from both sides. The root was guided to growstraight along the narrow channel in the front compartment of
the culture vessel to reduce the error by deviation in the image
depth. The channel length was 50 mm and cross sectional
dimensions of the channel were 3 mm × 2 mm. Photo imageof the seedling in the culture vessel is shown in Figure 2.
2.2. Structure of magnified time lapse image capture system
The root growth was recorded by the magnified time lapse
image capture system. The schematic of the system is shownin Figure 3. The image was captured by the Nikon1 J1digital camera, through the single focus macro lens,
Micro-Nikkor 105 mm F2.8, with 35 mm Microscopic Lens
24 × CM-3500 (Yoshida Industry Co., Ltd). The focal length
behind the 105 mm lens was extended by 55 mm with theextension lings, PK-11, PK-12 and PK-13 for larger
magnification. This lens system can project the image area ofas small as 2 mm × 3 mm on the 8.8 mm × 13.2 mm size
CMOS image sensor, with 3872 × 2592 pixel resolution. The
aperture was fixed to 16 for larger focus depth, and shutter
speed was 1/40 second for an image capture. The shutter was
relayed automatically with programed intervals. Images were
captured with 1 minutes interval for over 120 minutes.

Fig. 3. Schematic of the Magnified time lapse image capture system.
Culture vessel and optics were fixed on the optical stage. Focus
and horizontal position of image was adjusted by the XY stage
while the vertical position was adjusted by precision jack. The
root of the specimen was illuminated by LED ring light around theclose up lens.

Average root growth rate of R. sativus is around 0.5 mm/hour,
so the root tip will be well within the image area of the systemfor 2 or 3 hours. ISO sensitivity was fixed to 800. A LED
ring light was equipped around the close-up lens to illuminate
the root specimen. The illuminance of the ring LED was ca.
3000 lx on the observation window.
The camera optics and the culture vessel were fixed on a
optical stage to reduce the mechanical vibration. Largedisplacement in vertical axis, by the growth of root, wasadjusted by the jacks under the vessel. Fine position invertical and horizontal axis were adjusted by the precision lab
jack and xy stage. Focusing to the root tip position was alsoenabled by the xy stage because of the narrow focusing rangeof the lens system, with the macro lens and the extension rings,
needs fine adjestment.
Figure 4 shows a image of the root tip of a specimen,
captured by the lens system. The seedling was inserted in a
cotton gauze strip which was fixed on the bottom of the acrylic
pocket on top of the culture vessel. The seedling was tightly
connected with the cotton gauze by root hair, so the boundary
point of root and stem was fixed to the vessel. The camera
was aligned to focus on the root tip by precision stages, andboth the vessel and the camera optics were fixed on a opticalbench. Relative position of the root base to the image was
fixed during the measurement, so the differences of root tip
position in the successive time lapse images are the root
growth within the shatter interval. The root length wasmeasured by a image processing program, Image J (Rasband,1997). The root length in a image was measured along thecenter of the thick white line, which is fitted to the curvature of
the root. The ratio of actual length and pixel numbers was
defined by measuring the pixel numbers of a image of a microruler for micro scope. The length/pixel conversion ratio wasFig. 4. A magnified image of a growing root of R. sativus seedling.
Root length was measured by Image J image processing software.
Thick white line is fitted to the curvature of the root image, androot length was measured along the center line.

used for calculation of root length by Image J.
2.3. Application of electric field to the root of water
cultured seedling
50 Hz a.c. electric field was applied to the root of seedlings
by a pair of platinum wires, located on top and bottom of thefront compartment of the culture vessel, 50 mm apart eachother. The thickness of the compartment is 2 mm, andelectric field is confined in the thin sheet of culture medium, so
the field intensity can be well approximated by dividing theapplied voltage by the electrode gap. 0 to 1500 V/m electricfield, with 300 V/m interval, was applied to the culture
medium with heating of less than 1˚C in the air conditioned
environment for the low conductivity of the medium.
Electric field was applied with the 5 minutes ON and OFFinterval pairs. In preliminary experiments, shorter interval
stimulation, such as 1 minute, failed to make effects within theinterval time and electric field effect was not clearly correlated
to the stimulation. Each session for one value of field
intensity consists of 3 pairs of ON/OFF switching, which takes
30 minutes. 5 sessions of field application, with increasing
field intensity of 100-500 V/m or 300-1500 V/m, are repeated
for a seedling specimen. Including the final 5 minutes 0 V/m
field interval, one experimental procedure takes 155 minutes.
For continuous growth rate measurement, all the images,
captured with 1 minute interval, were processed for growth ratemeasurement and evaluation of system resolution. However,in electric field application experiments, field applicationinterval and response time seems to be larger than 5 minutes.
And for slower growth rate of some specimen, only the imagesof 5 minutes interval were processed for data analysis.

3. Results and Discussion
3.1. Resolution of the image capture optics
Figure 5 (a) shows a image of a 2 μm diameter latex bead
(Fluoresbright microsphere Y2.0, Polyscience Inc.), taken by

(a) Optical image of latex bead, captured by the lens system. Brightnessof the image was measured along the horizontal line.
Fig. 6. Root growth of R. sativus without application of electric field.
3 specimens were cultured independently in the vessel and imagesof the root were captured over the range of 130 minutes withinterval of 1minutes. Specimen 1 and 3 showed change of root
growth rate around the 60-100 minutes after the start of themeasurement.

(b) Brightness profile of the latex bead image. The full width at half
maximum was measured as about 4 μm.
Fig. 5. Resolution of the magnified time lapse image capture system.
For calibration of the resolution, images of latex micro bead with
2 μm diameter were captured and numerically analyzed.

the image capture system for calibration of optical resolution.
Brightness profile of the picture along the horizontal line is also
shown in Figure 5 (b). Full width at the half maximum(FWHM) was 4.3 µm. This result suggests that the
maximum resolution of less than 4 µm. The value of FWHM
was twice larger than the diameter of the microsphere.
Theoretical resolution of this system, calculated from the pixel
number of image sensor and image area, is 0.77 µm for a pixel.
However optical limit by interference of light, wave length
dispersion of refractive index and optical aberrations of the lenssystem degraded the resolution.
3.2. Deviation of root growth rate in short and long term
The root growth curves of 3 R. sativus specimens, measured
by this system over 135 minutes, are shown in Figure 6. The
vertical axis is the root length diferrence from the start ofobservation. Root lengths of the 3 seedlings were measured
with same condition. The lengths of the roots were measured
at every 1 minute. The individual growth rates of 3
specimens were differed each other in 135 minutes
measurement time, but range was within the 8-10 µm/min.
The increase of root length even in 1 minute can be
distinguished by this system. These results demonstrate theresolution and short time measurement capability of this system.

Fig. 7. Standard deviation of growth rate along the time course of
growth measurement. Slope of growth curve was calculated
for different numbers of data points, 3, 5, 9 and 15, over the total
data of 130 minutes.

While the root growth of specimen 2 was linear to the time,
the root growth rate of specimen 1 and 3 changed at around60-100 minutes after the start of the measurement. The
reason of these changes of growth rate is not determined. To
evaluate the growth rate deviation from the linear relation,slope of auto regression line was calculated for moving
window of 3, 5, 9 and 15 data over the time range of 10 to 125
minutes. The slope values were assumed as the root growthrate within the short time period. The fluctuation of thisgrowth rate is represented by the standard deviation of theseslope values over the 2 hours of observation time. The graphs
in Figure 7 show the standard deviations of these root growth
rates for the data window length of 3 - 15 data (#N in the
figure). One data correspond to 1 minute time span. The
standard deviation of root growth rate is the highest in 3
minutes time window and decreases with the length of the time
window. Standard deviation of each specimen decreased
with the width of data window, as is predicted by theoreticalconsideration about the relation of sample number anddeviation. However, decrease of standard deviation is notsignificant for the time window lengths longer than 5, so
minimum observation time window for this system wasassured as 5 minutes.
On the other hand, base lines of the 3 graphs in Figure 7
differ each other, because of individual difference of each seed
and long term shift of growth rate. As can be seen in thegraphs in Figure 6, growth rate of specimen 1 and 3 shifted intime range of some 10 minutes at around 60 minutes from thestart. These phenomena suggest the existence of long term
response of a plant to some environmental change. But weare not sure what caused these shift in the experiment for now.
At least, it can be concluded that root growth rate of a plantseedling can be changed in as short as some 10 minutes range.
These results show that observation with interval of longer thansome 10 minutes may fail to detect some plant response.
However, deviation by measurement error is so large in 3
minutes. From these results, the optimum time window forroot growth rate measurement is in the range of 5 to some 10
minutes.
3.3. The root growth under application of electric field
Figure 8 shows the root growth rate of one water cultured
seedling under application of electric field. The applied field
intensity is shown by the black rectangle column. The a.c.
electric field of up to 1500 V/m was applied in the culture
medium. Electric field was applied only in increasing orderbecause higher field intensity may cause some irreversible
change of plant tissue. With application of 300 V/m field,
growth rate gradually increased from 3 µm/min to 7 µm/min in
30 minutes. The increasing curve didn’t changed by ON and
OFF of the field, so the response time of growth enhancement
mechanism is speculated to be longer than 5 minutes. By
application of higher intensity field, however, growth rate was
reduced from 7 µm/min to 5-3 µm/min. This result suggests
the existence of some mechanism of growth inhibition by theelectric field of higher than 600 V/m. The response time ofthis mechanism seems to be shorter than 5 minutes because thegrowth rate was reduced by application of the field and slightly
recovered by removal of the field within 5 minutes interval.
For more precise investigation of field intensity, root
growth rate was also measured with application of lower
intensity field, 0-500 V/m, as shown in Figure 9. Root
growth rate was 9.5 µm/min from the start of observation.
100 V/m field seem to have little enhancing effect of root
growth but it attained the maximum growth rate of 11 µm/min
at 30 minutes, and the growth rate decreased by application ofhigher electric field. Over all growth rate was higher than 7µm/min, This value is almost same as the maximum growthrate in Figure 8. It is possible that the maximum growth rate
is limited by physiological reasons, and electric enhancement

Fig. 8. Root growth rate under application of 0-1500 V/m a.c. electricfield. 50 Hz a.c. electric field was applied on single seedlingspecimen repeatedly. The electric field was switched ON/OFF
with 5 minutes interval. Open rectangle columns show theintensity of applied electric field, scaled in right axis. Root growthrate in the interval is shown by symbols. The + symbol
corresponds to the 0 electric field interval. Other symbols
correspond to the different field intensities.

Fig. 9. Root growth rate under application of 0-500 V/m, 50 Hz a.c.
electric field. 0-500 V/m electric field was applied to anothersingle seedling specimen. Field intensity was reduced to 1/3 ofthe experiment in Figure 8 for precise investigation of filed
intensity dependence. Over all growth rate is larger than that of
the 300 V/m session in 0-1500 V/m experiment.

of growth rate from higher value is restrained within the
maximum. This means the electrical growth enhancement
may be applicable only for the seedling of lower growth rate.
The difference of initial growth rate of individual seedlings
might have disturbed the obvious change by electric field.
The cause of individual difference is discussed later.
All the results suggest the existence of 2 different
mechanism of electric effect on the root growth. One
mechanism has suppressing effect on the root growth withshort response time. This effect is negligible for the electricfield intensity of lower than 600 V/m. Another mechanism
has enhancing effect on the root growth with response time of
longer than at least 5 minutes. This mechanism is effective
within the range of 100-300 V/m. Maximum root growthrate of R. sativus seedling, within the experimental conditions,is about 11 µm/min and electric mechanism cannot overridethis limitation. Growth rates of 3 specimens in Figure 6 weredifferent, and even shifted in 2 hour experiment. Thisinstability of growth rate disturbed the consistent experimental
results. One possible reason of this instability is allelopathyof R. sativus seedlings. R. sativus seeds show lateralsuppression in germination phase (Suzuki et al., 2002). Sucha lateral control is generally observed in ecological systems.
To investigate this assumption and have stable results in culture
experiments, we need some modification of the culture vessel
for multiple cultivation of seedlings.
The root growth suppression by electric field has short
response time, and its mechanism may well be the electric
break down of some biochemical signaling system on the cell
membrane, such as ion channel or electrochemical receptor.
On the other hand, electric root growth enhancement seem tohave longer response time of some 10 minutes, which suggests
the participation of biochemical process. This mechanism isstill to be understood by the results in this study. One possible
target of electric stimulation is some electric receptor on thecell membrane. Some plant species are known to haveelectric potential around it’s root (Iwabuchi, 1989). Thispotential is speculated to be generated by the release of proton
by H+-ATPase (Suzuki et al., 2002). Proton concentration is
also closely related with the growth control of plant root
(Mentze et al., 1977; Toko et al., 1987). The function of thepotential is not well established so far. But if the electric
potential of proton gradient contributes to the growth control ofroot, weak electric field can cause some perturbation on thegrowth signal.
This speculation leads to the possibility of new electric
stimulation scheme of plant root in arid land. Especially insalinated soil area, electric conductivity of bulk soil is rather
high. Electric current will make largest voltage drop at thehigh resistance of root surface or cell membrane, where theplant may be most sensitive to the electric potential change.
Electric stimulation, by installing stimulation electrode to thexylem of the plant and grounding the counter electrode to thesoil, will enhance root growth in high conductivity soil area.
Especially in this study, ion strength was restricted rather low
to avoid the Joule loss heating by higher field intensity. The
ion uptake is an important factor for the growth of R. sativus
seedling and the lower ion strength might have suppressed theroot growth. If the electric field of as weak as 100 V/m has aenhancing effect on the root growth rate, culture medium of
higher ion strength can be available. Precise design of
medium or soil conductivity, intensity and frequency of electricfield will create a new possibility of this system.

4. Conclusion
The magnified time lapse image capture system wasconstructed for the study of root growth enhancement by a.c.
electric field. The root growth enhancement of water cultured
R. sativus seedling by application of electric field wassuggested with this system.

References
Brayman A.A., Miller M.W. (1986): Induction of ELF
transmembrane potentials in relation to power-frequency
electric field bioeffects in a plant root model system.
Radiation. Environ. Biophys., 25: 141-149.
Hotta T., Suzuki S., Takahashi T. (2012): Investigation of The
Ion Concentration Effect on Eucalyptus Root Growth in a.c.
Electric Field. Proc. BMEiCON2012: 59-62.
Iwabuchi A., Yano M., Shimizu H. (1989): Development ofExtracellular Electric Pattern Around Lepidium Roots.
Protoplasma, 148: 94-100.
Mentze J. et al. (1977): Auxin-induced H+ Secretion in
Helianthus and Its Implications. Plant Physiol., 60: 509-512.
Rasband W.S. (1997-2014): ImageJ. U. S. National Institutesof Health, Bethesda, Maryland, USA, http://imagej.nih.
gov/ij/.
Suzuki S., Maruta T., Nakamura Y. (2001): The Effects ofElectric Field and lon Density on Root Growth. J. Faculty
Engng. Seikei Univ., 38(2): 1-8 (in Japanease).
Suzuki S., Maruta T., Nakamura Y. (2002): The Effect ofMetal ion in The Medium on the Growth of Rahpanus
sativus Seed and Lateral Inhibition. IEEJ Trans. C, 122:
1686-1691 (in Japanease).
Tanouchi H., Aikawa S., Kawasaki S., Utsugi H., Hamano H.,
Saito M., Adrianne K., Kojima T., Yamada K. (2008):
Examination of Biomas Production by Afforestation
Technology in Arid Land of Low-Latitude Zone. J.
Echotech. Research, 14(3): 183-188.
Toko K., Iiyama S., Tanaka C., Hayashi K., Yamafuji K.,
Yamafuji K. (1987): Ralation of Growth Process to Spacila
Patterns of Electric Potential and Enzyme aActivity in Bean
Roots. Biohyis. Chem., 20: 39-58.
Weisenseel M.H., Dorn A., Jaffe L.F. (1979): Natural H+
current traverse growing roots and root hairs of Barley. Plant
Physiol., 64: 512-51.