EFFECT OF ELECTROPORATION ON INTRACELLULAR ICE FORMATION IN RYE LEAF PROTOPLASTS

Cryomicroscopy was used to study the statistical behavior of intracell ular ice formation (IIF) in protoplasts isolated from rye leaves (Seca le cereale L cv. Aroostook) and subjected to prior electrical treatmen t (''electroporation''). Protoplasts were suspended in isotonic (530 m Osm) or hypotonic (265 mOsm) saline and exposed to exponentially decay ing electric fields of 50 to 500 V/cm and capacitances of 1 x 850, 2 x 850, or 3 x 850 mu F. Treatment conditions were selected by determini ng apparent cell lysis, viability, and increased permeability to fluor escent dyes. For each condition, protoplasts were subjected to linear cooling at 5, 10, 20, or 40 degrees C/min, or to cooling at 40 degrees C/min followed by an isothermal hold at -6, -9, -12, or -15 degrees C . In linear cooling, field strengths above 150 V/cm and capacitances a bove 1 x 850 mu F resulted in a 1 to 9 degrees C shift to higher media n mF temperatures. Maximum ITF incidence was unaffected. Under the wea kest condition (100 V/cm, 1 x 850 mu F), median IIF temperatures durin g linear cooling were shifted 3 to 7 degrees C below those of the cont rol. In isothermal periods, mean IIF times of electroporated protoplas ts were 50 to 300% smaller than the control, implying faster IIF kinet ics in electroporated protoplasts. In hypotonic saline the effects of electroporation were suppressed. Based on a comparison of stochastic m odel parameters, electroporation affected IIF during linear cooling an d isothermal periods similarly. (C) 1995 Academic Press, Inc.