OVERVIEW OF TECHNIQUES TO PREPARE LIGHT MICROSCOPIC MOUNTS OF <i>HELIANTHUS</i> (ASTERACEAE) REPRODUCTIVE STRUCTURES

Abstract

There are many investigations concerning material processing and staining of permanent or temporal mounts for light microscopy, and there are many works on Helianthus embryology either with the description of the methods or without it. Our aim was to make a review of these methods and to customize existing methods of permanent and temporal mounts staining for the work with representatives of the genus Helianthus to get good quality photo images.Wild perennial species Helianthus ciliaris, H. tuberosus, H. maximilianii, as well as cultivated forms of annual sunflower H. annuus were included in our research. The most of methods were worked out on H. ciliaris, and then were successfully used in the work with other Helianthus species listed above.When following classical methods, some stains become very pale or wash out completely during processing of the sunflower mounts. We developed some practices to avoid these failures. They are: prolongation of stains exposure; staining at higher temperature; reducing the time of processing by ethanol, acetic acid and other substances that can wash out the stain; changing the order of stains exposure; changing the way of mounts dehydration (drying in thermostat instead of treatment by absolute ethanol).We found out optimal techniques for staining of male and female reproductive structures of sunflower and the methods suitable for all structures: toluidine blue, Schiff’s reactive with alcian or toluidine blue as counter-stains, safranin with alcian blue. It is convenient to use acetocarmine solution for the estimation of sunflower pollen viability.Chloral hydrate, methyl benzoate or “Bio Clear” solution are applicable reagents for clearing ovules. The material after chloral hydrate or methyl benzoate is satisfactory to embed in paraffin and to make permanent mounts.