Preparation of enzymatically cleaned and isolated stomata

Preparation of enzymatically cleaned leaf epidermal strips of Nicotiana glauca

by Hudson S.,  Trail J., Simmons D., Baldwin L., Myers R., Tom B., Mohr J., Tallman G. (1983)

Stephen Hudson, Jeff Trail, Dwayne Simmons, Larry Baldwin, Rebecca Myers, Barry Tom, Judy Mohr, Gary Tallman, 

Natural Science Division, Pepperdine University, Malibu, CA 90265 U.S.A.

In  Plant Sci Lett 32: 1-8 – https://doi.org/10.1016/0304-4211(83)90092- 5 – 

https://www.sciencedirect.com/science/article/pii/0304421183900925

Abstract

A technique for enzymatically cleaning leaf epidermal strips of Nicotiana glauca so that guard cells remain the only intact and viable cells is described.

The method relies on differential sensitivities of epidermal, mesophyll, and guard cell walls to digestion with a mixture of Cellulysin and hemicellulase.

Guard cells isolated by this procedure remain joined at their ends as duplexes, retain continuous cell walls, and are free of epidermal and mesophyll cell wall fragments and cytoplasmic debris. Yields range from 90–95% of original cells. All recovered cells concentrate neutral red and exclude trypan blue. In addition, duplexes isolated from either adaxial or abaxial surfaces show significant increases in width and aperture over dark controls when illuminated in solutions containing 30 mM KCl. Yields approach those necessary for standard methods of biochemical analysis. A brief comparison with existing techniques for guard cell isolation is included.