Structural Preservation and Retention of Enzymatic Activity in Quenched, Lyophilized Plant Tissue

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Abstract
Maintenance of structural integrity and enzymatic retention were investigated in plant [Hebe buxifolia and Dianthus chinensis] tissue that was quenched and lyophilized by several procedures. Structural preservation of tissue, whole and sectioned, was evaluated by scanning electron microscopy and compared with the results obtained from chemically fixed, critical point-dried material. Whole shoot tips quenched in a liquid N slurry followed by drying in a fabricated lyophilizer were damaged the least, showing collapse of some epidermal cells. Quenching in dry ice-acetone or liquid N, followed by drying in a commercial lyophilizer, resulted in severe ice damage or torsion, respectively. The surface of quenched, lyophilized cryostat sections, compared with chemically fixed ones, appeared to melt during the sectioning process. Enzymatic retention was tested by a comparison of G-6-P dehydrogenase (G6PdH) activity in fresh vs. quenched (liquid N slurry), dried (fabricated lyophilizer) leaf tissue. The 2 tissue preparations demonstrated corresponding activity of G6PdH, measured fluorometrically. The presence of this same enzyme in fresh leaf tissue was verified by light microscopic histochemical staining of cryostat sectioned leaves. Evidently, quenching in a liquid N slurry followed by drying in a fabricated lyophilizer gave optimal results with regard to structural preservation and retention of enzymatic activity.