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United States Department of Agriculture

Agricultural Research Service

Research Project: EVALUATION AND IMPROVEMENT OF CEREAL GERMPLASM FOR DISEASE RESISTANCE AND WINTER-HARDINESS

Location: Plant Science Research

Title: Microwave Processing of Crowns from Winter Cereals for Light Microscopy.

Authors
item Livingston, David
item Tuong, Tan Duy
item Haigler, Candace - NORTH CAROLINA STATE UNIF
item Avci, U - NORTH CAROLINA STATE UNIV
item Tallury, Shyam - NORTH CAROLINA STATE UNIV

Submitted to: Journal of Microscopy
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 15, 2009
Publication Date: August 15, 2009
Citation: Livingston, D.P., Tuong, T.D., Haigler, C., Avci, U., Tallury, S. 2009. Microwave Processing of Crowns from Winter Cereals for Light Microscopy.. Journal of Microscopy. 49:1837-1842

Interpretive Summary: Traditional preparation of tissues for microscopy is time consuming because it requires a passive means of infusion of solvents into tissues. Referred to as "bench fixing", traditional methods can as long as 2 weeks before some samples can be sectioned for viewing. Microwave processing of tissues considerably shortens this time but there is not a “one-size-fits-all” procedure for microwave processing of plant tissue. None of the existing protocols for processing plant tissue worked on crown tissue from winter cereals. The crown is the lower part of the stem just above the root that is important for survival of the plant during winter. Existing protocols all caused crown tissue to shatter and fall apart when they were sliced into thin sections in preparation for looking at under a light microscope. This paper describes a protocol that was developed using microwaves that allows crown tissue to be processed in a single day instead of 2 weeks using traditional preparatory protocols. It was shown to work well for oat, barley, wheat and rye and will likely work with any grass species that has a crown similar to winter cereals.

Technical Abstract: Microwave processing of tissue considerably shortens the time it takes to prepare samples for light and electron microscopy. However, plant tissues from different species and different regions of the plant respond differently making it impossible to use a single protocol for all plant tissue. The crown of winter cereals is the below-ground portion of the stem that overwinters. It is composed of numerous types of cells with an organizational pattern that is unique to grasses. Microwave protocols developed for other plant tissues resulted in paraffin-embedded, winter-cereal crown tissue shattering and crumbling when mictotomed. This study reports a procedure developed to process winter cereal crowns from oat, wheat, barley and rye for histological observations. Using this protocol, crown samples were prepared in one day as compared to 2 weeks using traditional bench-fixing protocols. Results of varying time, temperature and microwave wattage during the 3 major divisions of tissue processing, namely fixing, dehydrating and embedding in paraffin are described. Results of sections from crowns of each of the 4 winter cereals indicate that this procedure is valid for crown tissue from all 4 species. Since the nature of crown tissue is similar across all grass species, it is likely that this procedure would work with the crown tissue of any grass species.

Last Modified: 4/16/2014
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