ARS | Publication request: A METHOD FOR DETERMINING LIPID DISTRIBUTION IN RICE GRAIN USING FLUORESCENCE MICROSCOPY

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: December 1, 2005
Publication Date: September 1, 2005
Citation: Wood, D.F., Cagampang, G., Matsler, A., Siebenmorgen, T. A method for determining lipid distribution in rice grain using fluorescence microscopy. International AACC Annual Meeting, September 11-14, 2005, Orlando, FL, Paper #350.

Technical Abstract: Standard plastic embedding methods from which high resolution sections can be produced for fluorescence microscopy include the use of chemicals in which lipids are solubilized. An alternative sectioning method in which lipids are preserved was developed in order to determine the location and distribution of storage lipids in rice grain sections. Whole rice grains were fixed in formalin - acetic acid - ethanol for several days and then stored in the fixative until they were prepared for sectioning. Individual grains were rinsed in distilled water and placed on a specimen mount using a water-soluble cryo “glue” containing polyvinyl alcohol and polyethylene glycol (Tissue-Tek). The specimen and mount was frozen in a Leica CM3000 cryostat microtome at –30 degrees (o) C. Blocks were faced and sectioned with a low profile blade at –20 degrees (o) C in the cryostat microtome. Adhesive tapes and adhesive-coated slides designed specifically for cryo microtomy were used (Instrumedics, Inc., Hackensack, NJ). An intact section was collected by placing adhesive tape onto the specimen face, holding the top of the tape with fingers and making the section. The adhesive side of the tape was placed on an adhesive-coated slide. The slide and section was removed from the cryostat, placed under UV light for 15 min to polymerize the adhesive on the slide. The tape was then gently removed from the slide and the slide was washed with distilled water to removed the OCT Compound. The section was stained with Nile Blue A or Oil Red O for lipid and observed in the fluorescence microscope. The method was applied to four rice cultivars and the lipid distribution was compared.