Regeneration of Freezing-Tolerant Spring Wheat (Triticum aestivum L.) Plants from Cryoselected Callus

Abstract

A cryoselection protocol has been developed that provides freezing-tolerant callus that, in turn, can regenerate plants with enhanced cold hardiness. Tolerant calli were selected from spring wheat (Triticum aestivum L.) callus by immersion in liquid nitrogen without addition of cryoprotectants. Less than 15% of the calli survived the initial challenge, whereas 30 to 40% of previously selected calli survived subsequent exposure. Seed progeny from five of 11 regenerant (R2) lines tested exhibited significantly enhanced tolerance to freezing at -120C. Thus, cryoselection appears to involve at least in part, selection for genetic rather than epigenetic variants. Analysis of one callus line indicated that cryoselection did not induce significant alterations in lipid composition, adenylate energy charge, or freezing point. An increase in the soluble sugar component was detected. Changes were also detected in the protein complement of microsomal membrane and soluble protein extracts of cryoselected callus. In all, seven unique proteins ranging from 79 to 149 kilodaltons were identified. The results demonstrate that freezing tolerant callus can be isolated from a heterogeneous population by cryoselection, and factors that contribute to hardiness at the callus level are biologically stable and can contribute to tolerance at the whole plant level.esses occur where extraorgan freezing and intracellular solute accumulation are important strategies for survival. Several reports indicate that, at least within a species, the relative water content of meristematic regions is an important factor in survival (6, 15). Both factors limit the availability of freezable water and therefore, reduce the likelihood of intracellular ice formation.It is clear that variability in response to freezing stress extends even to the level ofa single cell line (7). As an extreme example, in a previous study from this laboratory, Chen and associates (4, 5) reported that 7.2% of Catharanthus roseus and 2.5% of Triticum aestivum suspension cells survived immersion in liquid nitrogen without the addition of cryoprotectants. From this observation it was speculated that severe freezing stress might be used to isolate freezing-tolerant variants from a heterogeneous population of cells.The present investigation was undertaken to determine: the extent of survival of spring wheat callus following freezing in LN22 without the addition of cryoprotectants; the ability of such "cryoselected" callus to demonstrate enhanced LN2 freezing tolerance after a second challenge; the potential of the cryoselected callus to regenerate plants with enhanced (conventional) freezing tolerance; and the biochemical alterations, if any, that might correlate with survival.Plant species exhibit varying degrees oftolerance to freezing stress. For example, while in temperate grasses the minimum temperature for survival is in the -15°to -30°C range, some woody species, in the fully hardened state, can survive immersion in liquid nitrogen (12). This variability is also reflect...

In vitro conservation of tropical plant germplasm — a review

“…Similar results were observed with transformed roots of Beta vulgaris and Nicotiana rustica checked for their T-DNA structure (Benson & Hamill, 1991). However, in a recent work with Triticum aestivum, Kendall et al (1990) could select freezing tolerant calluses by repeated exposures to liquid nitrogen. The cryoselected calluses regenerated plants with enhanced cold hardiness.…”

Cryopreservation of isolated micropores of spring rapeseed (Brassica napus L.) for in vitro embryo production

“…This suggests that the survival of cells and tissues in liquid nitrogen is due to the physical alteration of cells and tissues by cryopreservation process rather than from a genetic selection. However, Kendall et al (1990) selected freezing tolerant callus of spring wheat (Triticum aestivum) which, in turn, can regenerate plants with enhanced cold hardiness. They speculated that freezing stress may have selected or induced a cell line genetically distinct from its progenitor.…”

Cryopreservation of Orchid Genetic Resources by Desiccation: A Case Study of Bletilla formosana

“…Pretreatment is critical and should be applied to most plant tissues before cryopreservation, as stated in the preceding paragraph. Therefore, although cryopreservation has been employed for the long-term preservation of plant tissues for a number of decades, investigation into pretreatment techniques has continued to contribute to improved tissue viability [14,34,47].…”