Protoplasts Surviving Freezing to −196 C and Osmotic Dehydration in 5 Molar Salt Solutions Prepared from the Bark of Winter Black Locust Trees

Abstract

Free protoplasts were prepared from the living bark tissue of the trunk of summer and winter black locust trees by enzymic digestion of thin slices of the tissue for 3 hours in a medium containing 2% Onozuka cellulase, 2% Rhozyme pectinase, and 2% Driselase in mannitol solutions using 0.4 molar mannitol for summer tissue and 1.0 molar mannitol for winter tissues.Cleaned suspensions of protoplasts and also thin slices of tissue with cells intact were frozen to temperatures of -10 C, -20 C, -30 C, -40 C and liquid nitrogen in sucrose and balanced salt solutions. Similar suspensions of protoplasts were also subjected to strong osmotic dehydration (plasmorrhysis) in a series of balanced salt solutions of increasing molarity. Tests for survival showed that protoplasts retain the same properties of either extreme susceptibility or extreme resistance to injury by freezing or osmotic dehydration as the cells from which they are prepared. Winter protoplasts showed capability for tolerating freezing to -196 C and plasmorrhysis in 5 molar salt solutions. These results indicate that protoplasts are a valid and useful system for investigating the properties of the protoplasm and surface membranes associated with the seasonal development of extreme hardiness in the cells of woody plants.Studies of freezing injury and resistance in plants increasingly point to the plasma membrane as the primary site of these phenomena (9)(10)(11)16). Free plant protoplasts afford a system for studying the cellular and biochemical properties of these membranes free of the complications associated with presence of cell walls (12, 15). The preparation of protoplasts from tissues of both unhardened and hardened plants which retain the original properties of freezing resistance of their parent cells has therefore been a target for investigators in this field. Such protoplast preparations have been made recently from moderately hardy winter cereals which are completely killed by freezing to -7 C in the unhardened condition and tolerant of -18 C in the hardened condition (1). These protoplasts have permitted studies that indicate the protoplasts not only behave like normal cells in freezing (13) but possess the same differences in degree of resistance to freezing and osmotic dehydration as their parent cells (7,14 membranes. The present paper describes the successful preparation of protoplasts from the bark of the winter and summer black locust trees which retain almost entirely the respective extremes of tolerance or susceptibility to freezing and osmotic dehydration of the cells and tissues from which they were prepared. MATERIALS AND METHODSThe living bark tissues were excised from the trunks of a group of 10-year-old black locust trees growing in the Ottawa area. Summer trees were sampled during the month of July, while winter tissues were obtained from thawed short lengths of trunk cut from the same group of trees in January and stored frozen at -10 C for 6 months. The cold hardiness of these frozen-stored logs was identical with that of...

Relationship between Phospholipid Breakdown and Freezing Injury in a Cell Wall-Less Mutant of Chlamydomonas reinhardii

“…Because the mutant CW15+ lacks the cell wall found in the wild-type, this indicates that disruption of the plasma membrane/cell wall interaction was not a primary cause of freezing injury in C. reinhardii. Similar conclusions have resulted from studies of isolated protoplasts of higher plant cells (19).…”

Antifreeze proteins are secreted by winter rye cells in suspension culture

“…However, it is not yet known whether AFPs also exert cryoprotective effects in planta as observed in experiments using fish AFPs. These experiments are difficult to conduct with intact plants; however, suspension cells and protoplasts isolated from non‐acclimated and cold‐acclimated winter rye, bromegrass and the woody species Robinia pseudoacacia have been shown to exhibit changes in freezing tolerance that are comparable with the corresponding tissues and organs (Siminovitch 1979, Singh 1981, Chen and Gusta 1982, Ishikawa et al 1995). One advantage of working with protoplasts and suspension cells is that externally applied solutions are more uniformly accessible to the cells.…”

Role of cell wall in freezing tolerance of cultured potato cells and their protoplasts

“…Siminoviteh et al (1978), however, observed a higher degree of freezing tolerance of protoplasts than of their parent tissue in rye seedlings. Siminovitch (1979) also reported a lower resistance to injury of protoplasts (of black locust bark) frozen in sucrose eompared to intact cells in tissue slices. Singh (1981) pointed out that, to prevent lysis, the released protoplasts require the presence of an external osmoticum.…”

“…Released protoplasts from higher plants have been used to study the mechanism of freezing toleranee and cold acclimation. The use of protoplasts not only allows microscopic observations of membrane ehanges (Steponkus et al 1982a), but faeilitates in situ labeling of the plasma membrane with probes (Singh 1979) and avoids the "complications associated with the presenee of eell wall" (Siminovitch 1979) in such studies. When released protoplasts were frozen in an appropriate medium, they exhibited freezing sensitivity similar to or parallel with their tissues of origin (Singh 1979, Steponkus et al 1982a.…”