Stable non-Gaussian random processes , by G. Samorodnitsky and M. S. Taqqu. Pp. 632. £49.50. 1994. ISBN 0-412-05171-0 (Chapman and Hall).

A model to predict global patterns in vegetation physiognomy was developed from physiological considera- tions influencing the distributions of different functional types of plant. Primary driving variables are mean coldest- month temperature, annual accumulated temeprature over 5"C, and a drought index incorporating the seasonality of precipitation and the available water capacity of the soil. The model predicts which plant types can occur in a given environment, and selects the potentially dominant types from among them. Biomes arise as combinations of domi- nant types. Global environmental data were supplied as monthly means of temperature, precipitation and sunshine (interpolated to a global 0.5" grid, with a lapse-rate correc-

A global biome model based on plant physiology and dominance, soil properties and climate

Cells can endure storage at low temperatures such as--196 degrees C for centuries. The challenge is to determine how they can survive both the cooling to such temperatures and the subsequent return to physiological conditions. A major factor is whether they freeze intracellularly. They do so if cooling is too rapid, because with rapid cooling insufficient cell water is removed osmotically to eliminate supercooling. Equations have been developed that describe the kinetics of this water loss and permit one to predict the likelihood of intracellular freezing as a function of cooling rate. Such predictions agree well with observations. Although the avoidance of intracellular freezing is usually necessary for survival, it is not sufficient. Slow freezing itself can be injurious. As ice forms outside the cell, the residual unfrozen medium forms channels of decreasing size and increasing solute concentration. The cells lie in the channels and shrink in osmotic response to the rising solute concentration. Prior theories have ascribed slow freezing injury to the concentration of solutes or the cell shrinkage. Recent experiments, however, indicate that the damage is due more to the decrease in the size of the unfrozen channels. This new view of the mechanism of slow freezing injury ought to facilitate the development of procedures for the preservation of complex assemblages of cells of biological, medical, and agricultural significance.

Freezing of living cells: mechanisms and implications

http://science.sciencemag.org/content/sci/168/3934/939.full.pdf

Cryobiology: The Freezing of Biological Systems

Numerous studies are reviewed of the plasma membrane in relation to freezing injury and cold acclimation. An attempt is made to explore the nature of freezing injury of plants on which to formulate a mechanism(s) of injury and how the biochemical changes occurring during cold acclimation can be accurately assessed. Although it is likely that all cellular membranes are susceptible to freezing injury, there has been a tendency to establish a hierarchy of injury to various cellular membranes. Nevertheless, it is the plasma membrane that plays a central role in the behavior of the a cell during a freeze-thaw cycle. 247 references.

Role of the Plasma Membrane in Freezing Injury and Cold Acclimation

This paper discusses the importance of the successive steps of the vitrification technique and reviews the current development and use of vitrification and of the two derived protocols, encapsulation-vitrification and droplet-vitrification Vitrification refers to the physical process by which a highly concentrated cryoprotective solution supercools to very low temperatures and finally solidifies into a metastable glass, without undergoing crystallization at a practical cooling rate Samples are thus cryopreserved without detrimental intracellular ice formation In a standard vitrification protocol, excised explants are precultured on medium enriched with sucrose, treated (loaded) with a loading solution composed of 2 M glycerol + 04 M sucrose, dehydrated with a highly concentrated vitrification solution [eg the PVS2 vitrification solution, which contains 30 percent (w/v) glycerol, 15 percent (w/v) ethylene glycol and 15 percent (w/v) DMSO and 04 M sucrose], frozen and rewarmed rapidly, unloaded with basal culture medium supplemented with 12 M sucrose, and then transferred to standard culture conditions In the encapsulation-vitrification technique, the explants are encapsulated in alginate beads, loaded and dehydrated with a vitrification solution before rapid immersion in liquid nitrogen In the droplet-freezing technique, excised explants are loaded, treated with the vitrification solution and frozen in individual microdroplets of vitrification solution placed on aluminium foils, which are immersed rapidly in liquid nitrogen These three techniques have been applied to different tissues of over 100 plant species from temperate and tropical origins and the number of cases where they are being tested on a large scale or applied routinely is increasing

Vitrification, encapsulation-vitrification and droplet-vitrification: a review.

The role of sugar and related compounds in variations of freezing resistance.

A simplified method for the isolation of a plasma membrane-enriched fraction from plants utilizing an aqueous two-polymer phase system is outlined. Mainly, the plant used was Orchard grass (Dactylis glomerata L.). The two-phase system consisted of 5.6% (w/w) of dextran T500 and 5.6% (w/w) of polyethyleneglycol 4000 in 0.5 molar sorbitol-15 millimolar Tris-maleate (pH 7.3), and 30 millimolar NaCl. In this system, the plasma membranes and the other membranes were preferentially partitioned into the top phase and into the lower phase, respectively. The purity of the isolated plasma membrane was sufficiently high even after a single partition (i.e. about 85% purity) and more than 90% purity was obtained after repeating the partition in a newly prepared lower phase. The plasma membrane was identified with the aid of phosphotungstic acid-chromic acid stain and the association of vanadate-sensitive Mg2+-ATPase. The plasma membrane-associated ATPase had a pH optimum at 6.5 and showed a high specificity for Mg2+ and ATP. KCl stimulation was low (6% stimulation) at the pH optimum, but a relatively high stimulation (23%) occurred at pH 5.5. This method for plasma membrane isolation may be applicable to a wide variety of plants and plant tissue including green leaves.

Partition of membrane particles in aqueous two-polymer phase system and its practical use for purification of plasma membranes from plants.

In vitro-grown shoot tips of apples (Malus domestica Borkh. cv. Fuji) were successfully cryopreserved by vitrification. Three-week-old in vitro apple plantlets were cold-hardened at 5°C for 3 weeks. Excised shoot tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 5°C. Following preculture shoot tips were transferred to a 2 ml plastic cryotube and a highly concentrated cryoprotective solution (designated PVS2) was then added at 25°C. The PVS2 contains (W/V) 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in medium containing 0.4 M sucrose. After dehydration at 25°C for 80 min, the shoot tips were directly plunged into liquid nitrogen. After rapid warming, the shoot tips were expelled into 2 ml of MS medium containing 1.2 M sucrose and then plated on agar MS medium. Direct shoot elongation was observed in approximately 3 weeks. The average rate of shoot formation was about 80%. This vitrification method was successfully applied to five apple species or cultivars and eight pear cultivars. This method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of fruit trees.

Akira Sakai

Papers

Vitrification, encapsulation-vitrification and droplet-vitrification: a review.

The role of sugar and related compounds in variations of freezing resistance.

Partition of membrane particles in aqueous two-polymer phase system and its practical use for purification of plasma membranes from plants.

Cryopreservation of in vitro -grown shoot tips of apple and pear by vitrification

Development of PVS-Based Vitrification and Encapsulation–Vitrification Protocols