Protoplasma 102, 343--347 (1980) PROTOPta SMA �9 by Springer-Verlag 1980

A M e t h o d f o r L o c a l i z i n g E m b r y o n a l L a t i c i f e r s b y C o m b i n e d

C o n v e n t i o n a l a n d F l u o r e s c e n c e M i c r o s c o p y ~

A. BI~UNI ': and BARBARA TOSI

Institute of Botany, University of Ferrara

Received October 23, 1979 Accepted November 4, 1979

S u m m a r y

The authors describe a simple method based on malachite green and acid fuchsin for the detection of laticifers during the embryogenesis of some Euphorbiaceae plants by conven- tional and fluorescence microscopy. The strong sensitivity and specificity of the method make it suitable for the ontogenetic studies of laticifers. The results obtained are discussed in the context of the reactive mechanism of the staining and of the chemical composition of the embryonal laticifers.

Keywords: Double staining; Euphorbiaceae; Fluorescent staining; Laticifers.

1. Introduct ion

Recently, great at tention has been devoted to the study of the latex com- position of Euphorbiaceae plants; first, for medical purposes due to the vescicant, irri tant, and co-carcinogenic properties in animal and human skin and mucous membranes (WATT and BREYER-BRANDWIJK 1962, MORTON 1971, KINGHOI~N and EVANS 1975, K~EI.ER et aI. 1978), and second, for purposes pertaining to marke t technology such as fish poison, insecticide and future resources of gasoline (MORTON 1971, BIJCHANAN et al. 1976). Despite this, the studies on the genesis and morphological organizat ion of the latex system represent a much neglected field of research (MAHImERC and SABHARWAI. 1968, BRUNI et al. 1978). The genesis of non-art iculated laticifers, the format ion of the latex system, and the correlation with the other tissues during growth and differentiat ion show very intricate problems, par t ia l ly due to the dif- ficulty of localizing the laticifers in the context of the embryonal tissues.

This paper was presented in part at the International Meeting on Botanical Microscopy, organized by The Royal Microscopical Society in York, July 9-13, 1979. ':" Correspondence and Reprints: Institute of Botany, University of Ferrara, Corso Porta Mare 2, 1-44100 Ferrara, Italy.

0033-183X/80/0102/0343/$ 01.00

344 A. BRt;NI and BAi~BARA TosI

We have devised a simple method, based on malachite green-acid fuchsin dyes, which has proved most efficient, both in conventional and fluorescence microscopy.

2. Materials and Methods

The plant material used was mature embryos of Euphorbia rnarginata, E. lucida, E. amygda- Ioides, and E. characias. The embryos, obtained by dissecting dry seeds, were fixed in a para- formaldehyde solution (10% w/v) in 0.2 M phosphate buffer at pH 7.4 for 1 hour, or in FAA (ethanol 70-glacial acetic acid-formalin, 9 : 5 : 5 ) for 24 hours. In both cases, the material was thoroughly washed after fixation, The embryos were dehydrated in graded ethanol and embedded in a mixture consisting of butyl-methyl methacrylates 7 : 3 (Merck, Darmstad't). After polymerization in a 50 ~ oven, the specimens were cut at 2-3 ~tm by an LKB Pyra- mitome with glass knives. Transversal and longitudinal sections were individually expanded in a drop of water on a clean slide. The plastic was removed by benzene, and the sections were immersed for 5 minutes each in absolute, 95%, 70~ and 50% alcohol. The specimens were stained by the following procedure, modified from PI•NESE (1896): a) staining with a solution composed of malachite green l~ aqueous-acid fuehsin 1~ aque- ous-distilled water 5 : 1 : 9 for at least 2 hours; b) rinsing of specimens by pouring a few drops of 95% alcohol over each slide, to wash away the excess dye; c) immersion for two minutes in 950/o alcohol and then treatment with absolute alcohol and xilene to clean; d) mounting in a rapid synthetic nonfluorescent medium (UV-inert, Serva, Heidelberg). As a control, specimens were observed unstained, or after only malachite green or acid fuchsin staining. All the preparations were examined and photographed with a Zeiss Photomicroscope II, equipped with an incident fluorescence condenser and a 75 Watt Xenon arc source. The band pass filter BP 546/10 was employed as a primary filter to give "green excitation", while a chromatic beam splitter FT 580 and longwave pass filter LP 590 proved to be optimal for these studies.

3. Resul t s

Results indicate that the proposed method offers several advantages, both for the facility in execution and for the inexpensive cost of the materials employed, when compared to the fluorescamine method (BRuNI et aI. 1977). By conventional microscopy, the embryonal laticifers are easily localizable by malachite green-acid fuchsin double stain. The cytoplasm matrix is coloured in a pale purple-red, while the small protein bodies, typical of the embryonal laticifers, and nuclei are dark green in colour (Fig. 1 a). The chromaticity of the dyes is good; malachite green, however, permits an easier detection of the laticifers since acid fuchsin only furnishes a weak purple contrast. The cells surrounding the laticifers react very poorly to the double staining, and only nuclei and some protein bodies appear weakly stained. With fluorescence microscopy, embryonal laticifers are easily detectable because of the strong red fluorescence emitted by the cytoplasmic matrix (Fig. 1 b). The nuclei are weakly fluorescent and difficult to localize. The protein bodies of laticifers, which are smaller (l/4-i/8) than those of other embryonal cells, are quite evident, but less fluorescent than the cytoplasmic matrix. Generally, in comparison with observations in "white light", the

A Method for Localizing Embryonal Laticifers 345

microscopical image formed under "green light" excitation has a higher contrast and also permits the detection of very small latex vessels. In addition, the brilliance of the image in fluorescence is reinforced because the remnant tissues of the embryo appear weakly fluorescent. The control reactions have shown that the unstained sections emit a pale autofluorescence which does not disturb the staining. The sections, stained

Fig. 1 a-b. Longitudinal section of an embryonal radicle of Euphorbia marginata showing the vascular iaticifers fixed in FAA and stained by the malachite green-acid fuchsin procedure. • a Photographed in "white light" by tungsten lamp. b Photographed under "green light" excitation by Xenon lamp

only with malachite green and observed by fluorescence microscope, confirm that this dye is not fluorescent with the excitation filter used in this work. The embryonal laticifers, stained only with acid fuchsin, emit a red fluores- cence similar to that engendered by the malachite green-acid fuchsin mixture, but the efficency of fluorescence is weaker. Concerning the fixative action which assumes great relevance in fluorescence microscopy (PEARSE 1972), both coagulant and non-coagulant fixative mix- tures may be employed, but the best results are obtained using the coagulant ones.

346 A. BRUNI and BARBARA TosI

4. Discussion

One advantage of the proposed histological method is that it is suitable for observations both in conventional and fluorescence microscopy. The uses of a combined techniques increases the probability of obtaining deducible in- formation for each specimen. The images produced by xenon lamp are of better quality when compared with those obtained by tungsten lamp: fluores- cence microscopy furnishes highly contrasted images in which fine details are also detectable. The red fluorescence of the specimens is due to the particular chromatic characteristics of acid fuchsin. Commercial acid fuchsins are a complex mixture of sulphonated derivatives of triphenylmethane compounds, and they are well known for their capability of acting as a base towards anionic or weakly cationic dyes (GuRR 1965). Whereas acid fuchsin acts as a complementary stain of malachite green in conventional light, it is the primary stain in fluorescence microscopy. Control reactions have shown that red fluorescence is stronger when acid fuchsin is used with malachite green dye. This evidence indicates that malachite green interacts with acid fuchsin enhancing its emission intensity. The mechanism of staining reaction may be explained as an interaction of the two stains by which acid fuchsin also links with tissue elements that have already been stained by malachite green. In this way, malachite green acts as a kind of mordant for the acid fuchsin, adding new reactive sites. This interaction is not evident in white light, but is strongly manifested under "green ex- citation". Malachite green and acid fuchsin are not specific dyes for certain chemical groups, but they are normally used in plant histology to differentiate primary and secondary cell walls. With fluorescence microscopy, their combined use may furnish a higher number of histological parameters particularly in tissues which are usually difficult to stain. In conclusion, red fluorescence emission of the embryonal laticifers under "green light" excitation is not caused by the presence of particular substances or chemical groups, but it results from the high amount of protein-carbo- hydrate complexes contained in the cytoplasm matrix of these internal secretory structures (BiauNI et al. 1977). The proposed method offers advan- tages in procedure and outlay and, therefore, is very convenient to use in routine work. Furthermore, the strong fluorescence and its resistence to fading permit the use of low speed film, normally difficult to employ in fluorescence microscopy.

Acknowledgements

The authors are grateful to the Italian National Research Council (CNR) for financial support.

A Method for Localizing Embryonal Laticifers 347

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