Gas exchange measurement as a non-destructive viability assay for frozen-thawed, winter-dormant apple buds.

Low temperature studies with winter-dormant buds are severely limited by the lack of a rapid, non-destructive assay for their viability. Investigations involving the winter harvest of ecodormant buds of woody subjects, including cryopreservation, are restricted if viability cannot be assessed until dormancy is broken. If post-treatment grafting indicates low survival of the harvested population then further collection and study has to be delayed until the next winter season. This study trials the use of a portable gas exchange system able to discriminate between live and dead buds rapidly, with the assay confirmed as non-destructive by subsequent micropropagation. Active respiration was recorded for 85% of a winter-dormant Malus domestica buds population that showed 91% viability when grafted (n = 45). Lethally stressed material gave no false positive results. When micropropagated after respiratory measurement, a population viability of 76% was recorded. There was a significant, positive correlation between respiration and fresh weight for buds of mass >10 mg, from a population with a mean fresh weight of 17 mg.

Cryopreservation of winter-dormant apple: iii - bud water status and survival after cooling to -30°C and during recovery from cryopreservation.

In a continuing study to improve the efficiency of dormant bud cryopreservation for tissues hardened in maritime climates, the water status of dormant buds was monitored between -4 degree C and recovery from liquid nitrogen (LN). Measurement of water content, simple thermal analysis and differential scanning calorimetry were employed. Buds did not lose water during cooling to, or holding at -30 degree C indicating that cryodehydration and/or other adaptive responses contributed during this essential step. A bud exotherm that was an artefact of warming was detected due to necessary handling at -4 degree C before cooling to -30 degree C. There were no significant differences between cultivars with respect to water status at -30 degree C or immediately upon rewarming from LN despite significant differences in post-LN survival. Buds rehydrated in 5 days, but up to 14 days may be needed for recovery for some cultivars. In some instances buds could be grafted without rehydration, taking up water across the early graft union.

Cryopreservation of winter-dormant apple buds: I -Variation in recovery with cultivar and winter conditions.

The widely-adopted protocol for the cryopreservation of winter buds of fruit trees, such as Malus and Pyrus, was developed in a region with a continental climate, that provides relatively hard winters with a consequent effect on adaptive plant hardiness. In this study the protocol was evaluated in a typical maritime climate (eastern Denmark) where milder winters can be expected. The survival over two winters was evaluated, looking at variation between seasons and cultivars together with the progressive reduction in survival due to individual steps in the protocol. The study confirms that under such conditions significant variation in survival can be expected and that an extended period of imposed dehydration at -4 degree C is critical for bud survival. The occurrence of freezing events during this treatment suggests that cryodehydration may be involved, as well as evaporative water loss. To optimize the protocol for maritime environments, further investigation into the water status of the explants during cryopreservation is proposed.

Cryopreservation of winter-dormant apple buds: II - tissue water status after desiccation at -4C and before further cooling.

The established protocol for the cryopreservation of winter-dormant Malus buds requires that stem explants, containing a single, dormant bud are desiccated at -4 degree C, for up to 14 days, to reduce their water content to 25-30 percent of fresh weight. Using three apple cultivars, with known differences in response to cryopreservation, the pattern of evaporative water loss has been characterised, including early freezing events in the bud and cortical tissues that allow further desiccation by water migration to extracellular ice. There were no significant differences between cultivars in this respect or in the proportions of tissue water lost during the desiccation process. Differential Scanning Calorimetry (to -90 degree C) of intact buds indicated that bud tissues of the cultivar with the poorest response to cryopreservation had the highest residual water content at the end of the desiccation process and froze at the highest temperature.

Contaminated liquid nitrogen vapour as a risk factor in pathogen transfer.

Liquid nitrogen in storage containers will gather particulate matter from the atmosphere, or the surfaces of containers placed into it, with time. Some of these accumulating particles may be pathogenic organisms and it can be demonstrated that their viability may be conserved by immersion in the liquid nitrogen. This contamination constitutes a risk to the status of stored samples that can, largely, be avoided by the use of appropriate techniques for sealing sample containers and sterilizing their outer surfaces. The present study uses fungal spores and organic crystals to demonstrate that such particles contained in liquid nitrogen are released back into the environment when nitrogen vapour cools programmable freezers or dry shippers. This demonstrates that storage in the vapour phase above liquid nitrogen still carries a real risk of sample, or facility, contamination. Regardless of the safety of the stored sample, this is a potential source of cross-contamination between repositories or experimental sites, both locally and internationally, that could have serious consequences in clinical and agricultural situations. This study provides evidence to suggest that this possibility, as yet unquantified, should be included in risk analysis of storage protocols for reproductive materials. The risk becomes diverse when, for example, semen and embryos are frozen at an agricultural site and the dry shipper can co-transport spores of contaminating crop plant pathogens to the destination site.

Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation.

The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. An alternative mechanism has been proposed by Morris (2006) that cell damage is a result of an osmotic imbalance encountered during thawing. This paper examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. Horse spermatozoa were cooled at a range of cooling rates from 0.3 to 3,000 degrees C/min in the presence of a cryoprotectant. The ultrastructure of the samples was examined by Cryo Scanning Electron Microscopy (CryoSEM) and freeze substitution, to determine whether intracellular ice formed and to examine alternative mechanisms of cell injury during rapid cooling. No intracellular ice formation was detected at any cooling rate. Differential scanning Calorimetry (DSC) was employed to examine the amount of ice formed at different rate of cooling. It is concluded that cell damage to horse spermatozoa, at cooling rates of up to 3,000 degrees C/min, is not caused by intracellular ice formation. Spermatozoa that have been cooled at high rates are subjected to an osmotic shock when they are thawed.

Cryopreservation of horse semen under laboratory and field conditions using a Stirling Cycle freezer.

A Stirling Cycle freezer has been developed as an alternative to conventional liquid nitrogen controlled rate freezers. Horse semen samples were cooled in 0.25 ml straws and 15 ml bags in the Stirling Cycle freezer under laboratory conditions and as a portable device, powered from a car battery. For comparison, straws were frozen in a conventional liquid nitrogen controlled rate freezer. Upon thawing, motility and viability of samples frozen in the Stirling Cycle freezer were not significantly different when compared to samples frozen in the liquid nitrogen freezer. Unlike liquid nitrogen systems, the Stirling Cycle freezer does not pose a contamination risk, can be used in sterile conditions and has no need for a constant supply of cryogen. The freezer has potential for use in veterinary and genetic conservation applications.

Unexpectedly high susceptibility of micropropagated rhubarb (Rheum rhaponticum L.) to spot disease caused by Ramularia rhei.

Rapid, large-scale micropropagation of a Rheum rhaponticum (rhubarb) breeding line, selected for the absence of autumn dormancy, produces changes typical of somaclonal variation. Among these is enhanced susceptibility to foliar infection by the spot disease pathogen Ramularia rhei, with consequential increased spore production on infected leaves. This provides an increased level of inoculum in the late season, leading to commercially unacceptable petiole spotting as spores are washed from the lamina on to the petiole surface. Infection of conventionally propagated plants does not exceed market acceptability. Consequently, to maintain the commercial value of this rhubarb selection micropropagation must be avoided, at least until an acceptably modified protocol is available.

Identification and characterization of pathogenic fungi causing spot disease on rhubarb (Rheum raponticum L.) in the UK.

This study reports on the acquisition of significant susceptibility of clonal rhubarb, produced by micropropagation, to leaf and petiole spot disease when compared to conventionally propagated material. This alteration in response to pathogens accompanies previously reported reductions in growth rate and poor winter survival. The two fungi isolated from lesions of both leaves and petioles have been identified as Ascochyta rhei and Romularia rhei, the former not previously having been reported in this role. Both fungi are able to cause spot infection of leaves and petioles of the micropropagated material. The effect of temperature on leaf infection from both fungi showed an optimum near 25 degrees C for A. rhei and close to 20 degrees C for R. rhei.