Demonstration of laser biospeckle method for speedy in vivo evaluation of plant-sound interactions with arugula

In recent years, it is becoming clearer that plant growth and its yield are affected by sound with certain sounds, such as seedling of corn directing itself toward the sound source and its ability to distinguish stuttering of larvae from other sounds. However, methods investigating the effects of sound on plants either take a long time or are destructive. Here, we propose using laser biospeckle, a non-destructive and non-contact technique, to investigate the activities of an arugula plant for sounds of different frequencies, namely, 0 Hz or control, 100 Hz, 1 kHz, 10 kHz, including rock and classical music. Laser biospeckles are generated when scattered light from biological tissues interfere, and the intensities of such speckles change in time, and these changes reflect changes in the scattering structures within the biological tissue. A leaf was illuminated by light from a laser light of wavelength 635 nm, and the biospeckles were recorded as a movie by a CMOS camera for 20 sec at 15 frames per second (fps). The temporal correlation between the frames was characterized by a parameter called biospeckle activity (BA)under the exposure to different sound stimuli of classical and rock music and single-frequency sound stimuli for 1min. There was a clear difference in BA between the control and other frequencies with BA for 100 Hz being closer to control, while at higher frequencies, BA was much lower, indicating a dependence of the activity on the frequency. As BA is related to changes from both the surface as well as from the internal structures of the leaf, LSM (laser scanning microscope) observations conducted to confirm the change in the internal structure revealed more than 5% transient change in stomatal size following exposure to one minute to high frequency sound of 10kHz that reverted within ten minutes. Our results demonstrate the potential of laser biospeckle to speedily monitor in vivo response of plants to sound stimuli and thus could be a possible screening tool for selecting appropriate frequency sounds to enhance or delay the activity of plants. (337 words).