Laser Confocal Scanning Microscopy and Transmission Electron Microscopy to Visualize the Site of Callose Fiber Elongation on a Single Conifer Protoplast Selected With a Micromanipulator

Abstract

We have developed a new method to observe the site of big and long spiral callose fiber elongation from a protoplast in liquid culture medium. Protoplasts of embryogenic cells of a conifer, Larix leptolepis, were cultured in the NH 4 NO 3 -free Murashige and Skoog's medium containing 50 mM MgCl 2 and 6% sucrose in a well in a 96-or 24-well culture plates. The protoplasts with elongated callose fibers were selected before fixation with glutaraldehyde, by picking up using a micromanipulator after electric treatment (DC 3 kV/cm) in the medium containing Alexafluor 488 phalloidin. Under laser confocal scanning microscopy (LCSM), two sites in the cell were stained clearly. One site was the nucleus and the other was the plasma membrane from which fibers elongated. Single cell transmission electron microscopy (TEM) was developed for observation of the microstructure at the site of fiber elongation on a single protoplast. A single protoplast which had a fiber was selected using a micromanipulator and transferred to an agarose bead at a low gelling temperature. The cells were fixed with cold glutaraldehyde and processed for TEM analysis. Elongated thin fibrils with vesicle-like structures could be observed by TEM.

Development of a method to measure mechanical properties of single elongated callose fibers in protoplast cultures of Larix leptolepis and Betula platyphylla

“…The method developed in this paper for measuring mechanical properties of callose fibers could be applied to different protoplast fibers. The callose fibers, which are developed in liquid protoplast cultures of several plant species, are easy to separate from the original protoplasts, when they were transferred to pure water or in medium containing fixation chemical, e. g. glutaraldehyde (Oyanagi et al, 2014). There is no deposition of callose on membrane of fiber-forming protoplast, as aniline blue dye stains only the fiber itself but not the surface of protoplast (Fukumoto et al, 2005).…”

“…Clarifying the mechanical properties of callose fiber might help for possible use as spiral microfilament material in micro-world for specific medicinal purpose (Oyanagi et al, 2014).…”

“…They were picked up and transferred to a medium or pure water in a well of a 4-well plastic dish (Nunc) or a 60 mm center-well organ culture dish (BD Falcon) by using a micromanipulator as previously described (Oyanagi et al, 2014;Sasamoto et al, 2003). The medium for Larix contained 50 mM MgCl 2 , while that for Betula contained 100 mM CaCl 2 or pure water to study the effect of a medium containing high concentrations of Ca 2+ ions.…”

“…Measurement of mechanical properties of unique callose fiber is an interesting topic, which might contribute to the development of an alkali-labile nano-machine at a cellular level (Oyanagi et al, 2014). Mechanical properties of single tomato suspension cell (Blewett et al, 2000) and large sized (several centimeters) discs of cell wall components including cellulose, were measured by compression test (Chanliaud et al, 2002).…”

New Factors for Protoplast-Callose-Fiber Formation in Salt-Tolerant Mangrove Plants, Avicennia alba and Bruguiera sexangula and Analysis of Fiber Substructures

“…Sub-structures of Betula protoplast-fibers were studied, though the protoplast culture methods were wrongly described (Seyama et al, 2008). The protoplast-fibers were studied further using laser confocal scanning microscopy (LCSM) with Alexafluor 488 phalloidin staining, and using single cell TEM to visualize the site of protoplat-fiber elongation from a single Larix protoplast selected with a micromanipulator (Oyanagi et al, 2014). Mechanical properties of single elongated protoplast-fibers of Larix and Betula were measured by a unique tensile test using two micromanipulators under an inverted microscope, and future development of an alkali-labile nanomachine at a cellular level was discussed (Oyanagi et al, 2014;2017).…”