Breeding Science 52 : 57-60 (2002)

NoteIntact Sectioning of Plant Tissues with a Cryomicrotome
Masahiko Maekawa*1) and Tadafumi Kawamoto2)
1)
2)

Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, JapanRI Research Institute, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi, Yokohama, Kanagawa 230-0063,
Japan

Key Words: cryomicrotome, thin frozen section, plants.

structure of the leaf was preserved as satisfactorily as that ofthe samples embedded with paraffin wax. The sections
showed clearly distinguishable cells and boundaries of green
sectors in the leaf tissues of the variegated albino plant(Fig. 1-B) which were found to be highly discriminative(Fig. 1-C, D and E). Moreover, it was observed that the chloroplast developed only in the green sectors (Fig. 1-C). Frozen sections, 3-20 µm thick, were obtained from plant
samples such as leaves and grains without any additional
manipulations. As a result, the method was also found to be
useful for thin-sectioning of plant tissues. We recommend
the use of an automatically operating cryomicrotome (LeicaCM3050S), though a manually operating one could be used,because cutting at a constant speed enables to obtain uniform specimens. Especially, for the sectioning of large-sized
samples, cutting at a constant speed is essential.
This method can also be applied for studies in variousfields, such as histology, general histochemistry, enzymehistochemistry, immunohistochemistry, in situ hybridization, autoradiography and electron microprobe X-ray microanalysis (Kawamoto and Shimizu 2000). For example, in thesignal transduction pathway, it is considered that calciumion plays an important role as a second messenger. The
actual mechanism still remains unknown because intact thinsectioned specimens of tissues to which X-ray microanalysis
was applied could not be obtained. Actually, Iwano et al.
(1999) observed drastic changes in the distributions of calcium ion in papillar cells and pollen grains in the process ofcross- and self-pollination in a self-incompatible species ofBrassica using an energy dispersive X-ray microanalysis
system. However, this observation was conducted only at
the surface of the stigma and it was difficult to observe thechanges in elemental distribution within the stigma. In thisregard, a intact sectioning method for X-ray microanalysis
may enable to obtain the mapping of the elemental distribution within a tissue directly.
Thus, we describe the application of the Kawamoto andShimizu method for the intact sectioning of plant tissues byusing a cryomicrotome.

In functional genomics as well as basic genomics, monitoring of gene expression in intact tissues provides various
kinds of useful information. In order to characterize mutant
phenotypes and conduct cellular analysis (Sylvester andRuzin 1994) as well as monitor gene expression in tissues,
thinner and uniform intact sections of tissues should beavailable. Although the use of a cryomicrotome which issuitable for preparing non-fixed thin sections was developed
half a century ago, it had been very difficult to obtain thinsectioned specimens of hard tissues or plant tissues.
Recently, Kawamoto and Shimizu (2000) have developed aconvenient system for obtaining uniform section of wholebody of a rat with completely calcified bone and teeth byusing a cryomicrotome. They used an adhesive film to avoid
damaging frozen sections during cutting. The film is specially prepared with synthetic adhesive (Cryo-film transferkit, FINETEC Co. Ltd. Japan) (Kawamoto 1990). Theirmethod could also be suitable for plant samples by using theadhesive film.
We tried to apply the method to plant samples and prepared intact frozen sections of rice leaves. In order to apply
the technique, the plant samples must be embedded withcarboxymethyl cellulose (CMC) gel. Since plants develop
a wax layer or trichome in the epidermis to protect themfrom rain or UV-B damage (Jansen et al. 1998), intact thinsectioning of plant tissues can not be easily achieved.
Parthasarathy (1994) reported that sucrose at appropriate
concentrations is very effective as a penetrating cryoprotectant for plant tissues. We conducted a pretreatment with a
10 % sucrose solution for sectioning rice leaves. Tween 20 asa surface-activating reagent was added to the solution enable
the solution to penetrate into the intracellular spaces of theleaves. By this treatment, the leaves could be well embeddedwith the CMC gel and cut.
As shown in Fig. 1, we obtained intact cross-sections ofrice leaves with variegated albino characteristics by combining the pretreatment and the use of an adhesive film. The

ProtocolCommunicated by K.Hattori
Received June 14, 2001. Accepted October 24, 2001.
*Corresponding author (e-mail: mmaekawa@rib.okayama-u.ac.jp)

Preparation of frozen sample block1) Cut the plant material of an appropriate length andtransfer the pieces to nylon mesh bags (Fig. 2).

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Maekawa and KawamotoFig. 1. Cross-section of un-fixed rice leaf (10 µm-thick). Normal (A) and variegated albino (C). B: variegated albino leaf before sectioning. D and E, Magnified view of the central portion and edge indicated by rectangles (d and e), respectively.

Fig. 2. Nylon mesh bag for gathering the samples and keeping
them in the solution.

2) Dip the mesh bags into a 10 % sucrose solution
with 0.3 % Tween 20 and hold them down thesewith stainless steel mesh (Fig. 3).
3) Put the sample in a low-pressure chamber for
10 minutes to remove air bubbles on the epidermis
and inject sucrose into the intracellular spaces.
4) Immerse the pieces into a stainless steel container
filled with 3-5 % CMC gel and place the pieces atthe bottom of the container (Fig. 4).

Fig. 3. Steel mesh in a beaker for keeping the samples in the solution in a low-pressure chamber.

Intact sectioning of plant tissues595) Dip the container into liquid nitrogen (or hexanedry ice) and freeze the pieces with the gel completely (take care not to splash the liquid into thecontainer in order to avoid cracks of the gel).
6) Take the frozen CMC gel out of the container.
7) Pour the CMC gel onto the sample stage of the cryomicrotome and place the frozen CMC gel on thesample stage.
8) Place the frozen sample on the cryomicrotome andtrim the sample with a disposable tungsten carbide
blade (Jung TC-65, Leica Instruments).

Fig. 4. Illustration of samples immersed
into CMC gel in a stainless steel container.

Preparation of adhesive film and sectioning
1) Spread 100 % EtOH on the acrylic board.
2) Place a polyvinylidene chloride film (10 µm thick;Asahikasei Kogyo, Japan) on the board uniformlyand squeeze out excess EtOH.
3) Apply the synthetic adhesive onto the film uniformly with a specially designed brush according
to a layout sheet for preparing the adhesive film
(Fig. 5).
4) After approximately 5 minutes, cut the adhesivecoated film into the size of the frozen CMC blockwith a rotary cutter.
5) Transfer the adhesive-coated film with the acrylic
board into the cryochamber.

Fig. 5. Illustration of application of synthetic adhesive on a polyvinylidene chloride
film on an acrylic board.

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Maekawa and Kawamoto6) Hold up a piece of the adhesive-coated film with aforceps and place the adhesive side of the film on
the trimmed surface.
7) Fix the film closely to the surface of the sample
with a soft cotton wad or paper.
8) Cut the sample slowly at a fixed speed (press gently the boundary between the blade and the sample
with a brush during cutting).
9) Hold up the film with the cut section and turn up
the sectioned side of the film on a glass slide in thecryochamber.
10) Add an aliquot of a 10 % sucrose solution containing 0.3 % Tween 20 onto the section and turn down
the sectioned side onto the glass slide.
11) Place the sectioned side of the film on the glass
slide from the edge slowly so as to squeeze out airbubbles.
12) Remove the excess solution with a filter paper.
13) Record the image of the section with a digital
camera.
14) Reproduce the image on a computer.

Acknowledgments
We thank Mr. K. Yamada, Leica Microsystems K.K.,
Mr. K. Ohe and the staff members of Osaka Sales Office,
FINETEC CO., LTD., for their technical assistance.

Literature Cited
Iwano, M., M. Wada, Y. Morita, H. Shiba, S. Takayama and A. Isogai(1999) X-ray microanalysis of papillar cells and pollen grainsin the pollination process in Brassica using a variable-pressure
scanning electron microscope. Jour. Elec. Micro. 48: 909-917.
Jansen, M.A.K., V. Gaba and B.M. Greenberg (1998) Higher plants andUV-B radiation: balancing damage, repair and acclimation.
Trends Plant Sci. 3: 131-135.
Kawamoto, T. and M. Shimizu (2000) A method for preparing 2- to 50µm-thick fresh-frozen sections of large samples and undecalcified hard tissues. Histochem. Cell Biol. 113: 331-339.
Kawamoto, T. (1990) Light microscopic autoradiography for study ofearly changes in the distribution of water-soluble materials. JHistochem Cytochem 38: 1805-1814.
Parthasarathy, M.V. (1994) Transmission electron microscopy: chemical fixation, freezing methods, and immunolocalization. In“The Maize Handbook” Freeling, M. and V. Walbot (eds.),
Springer-Verlag, New York. p. 118-134.
Sylvester, A.W. and S.E. Ruzin (1994) Light microscopy I: dissection
and microtechnique. In “The Maize Handbook” Freeling, M.
and V. Walbot (eds.), Springer-Verlag, New York. p. 83-95.