Novel perspectives on stomatal impressions: Rapid and non-invasive surface characterization of plant leaves by scanning electron microscopy

Abstract

Scanning electron microscopy (SEM) is widely used to investigate the surface morphology, and physiological state of plant leaves. Conventionally used methods for sample preparation are invasive, irreversible, require skill and expensive equipment, and are time and labor consuming. This study demonstrates a method to obtain in vivo surface information of plant leaves by imaging replicas with SEM that is rapid and non-invasive. Dental putty was applied to the leaves for 5 minutes and then removed. Replicas were then imaged with SEM and compared to fresh leaves, and leaves that were processed conventionally by chemical fixation, dehydration and critical point drying. The surface structure of leaves was well preserved on the replicas. The outline of epidermal as well as guard cells could be clearly distinguished enabling determination of stomatal density. Comparison of the dimensions of guard cells revealed that replicas did not differ from fresh leaves, while conventional sample preparation induced strong shrinkage (-40% in length and -38% in width) of the cells when compared to guard cells on fresh leaves. Tilting the replicas enabled clear measurement of stomatal aperture dimensions. Summing up, the major advantages of this method are that it is inexpensive, non-toxic, simple to apply, can be performed in the field, and that results on stomatal density and in vivo stomatal dimensions in 3D can be obtained in a few minutes.

Plant phenotyping relevance

植物葉の気孔密度・開口寸法を非侵襲的に取得するSEMレプリカ法を開発・比較検証しており、植物表現型取得法が中心である。

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