Martin, F. W.
While studying pollen tube growth in the styles of the wild Chilean tomato, L. chilense, it became desirable to stain the pollen tubes differentially with respect to the conducting tissue. A number of technics using vital stains were found by McGuire and Rick (1954) to be inadequate in this respect. The present method depends on the occurrence of callose, which lines and plugs pollen grains and pollen tubes. This technic is reliable, simple to use, and highly flexible with respect to time scheduling.
Styles to be observed are fixed in formalin-alcohol-acetic acid (F.A.A.) for 24 hours or more. After rinsing in tap water, the styles are treated in a nearly saturated aqueous solution of sodium hydroxide for 8 to 24 hours to clear and soften the tissue, permitting adequate penetration of the dye. The softened styles are transferred to a small beaker of tap water for one or more hours in order to remove most of the sodium hydroxide. Staining is accomplished next in a 0.1% solution of water-soluble aniline blue dye in 0.1 N K\3\PO\4 for 4 hours. Adequate staining can also be done in a small amount of the dye on a glass slide.
For observations the styles are mounted in a few drops of the staining medium on clean glass slides and are covered with cover slips. The styles are clear enough to be observed whole but may be neatly crushed by tapping the cover slip with a needle directly above the style. Observations are made with either a compound or a dissecting microscope by directly illuminating the slide with ultraviolet light of an approximate wavelength of 3560 Angstroms. An AH/4 100 Watt mercury lamp with appropriate UV filter was found to be satisfactory for this purpose. Such observations should be made only in a darkened room.
Under these conditions the callose in pollen tubes fluoresces bright yellow to yellow-green, whereas the background tissue fluoresces pale gray or blue. The amount and distribution of callose is highly variable. It sometimes nearly fills the tube making it visible for its entire length. In other cases the callose is confined to localized but closely spaced plugs. Occasionally the amount of callose in the tube is so small that portions of the tube, including the growing tip, are not visible. Varying amounts of callose occur frequently in the sieve tubes and in the epidermal hairs of the style, but with experience these deposits can usually be distinguished readily from the callose of the pollen tubes by their size, shape and distribution in the style.
Slides prepared in this manner may be sealed with wax or other media and stored for future use. Although preparations at room temperature begin to deteriorate within a few days, slides refrigerated at 40 deg. F will remain in good condition for one or two months.