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Background: Precise temperature control in several key areas during cryopreservation of dormant, winter apple buds is critical for maximal survival., Objective: To consider the effects of pre-harvest temperature, the duration of incubation at -30°C and variation in rewarming rate on survival., Materials and Methods: Dormant winter buds of Malus x domestica cultivars were harvested with two different acclimation histories and cryopreserved with variation in incubation time at -30°C. Recovery from LN using slow, intermediate and rapid rewarming was investigated as well as preservation after prolonged storage at -4°C., Results: The effects on survival of preharvest temperature regime and an altered -30°C incubation regime are cultivar dependent and an increase in rewarming rate has a strong negative effect on recovery., Conclusion: Post-thaw survival of the winter-dormant buds can be compromised by increased temperature over a period as short as 5 days prior to bud harvest. Varying incubation times at -30°C produce variable, cultivar dependent, survival and moderate increases in rewarming rates can also radically reduce survival.

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.

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.

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.

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.

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.

The cryopreservation of dedifferentiated cells, grown in suspension culture, is one of the portfolio of techniques employed for the long-term conservation of higher plant germplasm. Suspension cultures are also important in biotechnology, particularly in transformation studies and for the production of specific metabolites, and, here, there is also a pressing need for genetically stable, long-term storage of cell lines. Cryopreservation of suspension cell cultures can be exploited by either slow, or rapid, cooling techniques. During slow cooling the extracellular solutions are nucleated and the cells cryodehydrate during controlled cooling as a consequence of extracellular ice, to the point where their intracellular fluids will vitrify on subsequent transfer to liquid nitrogen. In the rapid cooling protocols, the cells are prepared by extreme osmotic dehydration, with cryoprotection, before plunging the samples directly into liquid nitrogen to achieve vitrification. Extensive success has been achieved with both techniques but rapid cooling is, currently, widely favored because of its simplicity.

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.

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.

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.

The essence of meristem-tip culture is the excision of the organized apex of the shoot from a selected donor plant for subsequent in vitro culture. The conditions of culture are regulated to allow only for organized outgrowth of the apex directly into a shoot, without the intervention of any adventitious organs (1-3). The excised meristem-tip is typically small (often less than 1 mm in length) and removed by sterile dissection under the microscope (Fig. 1). It comprises the apical dome and a limited number of the youngest leaf primordia, and excludes any differentiated provascular or vascular tissues. A major advantage of working with such a small explant is the potential that this holds for excluding pathogenic organisms present in the donor plant from the in vitro culture (see below). A second advantage is the genetic stability inherent in the technique, since plantlet production from adventitious organs can be avoided (3-7). However, if there is no requirement for such benefits, then the related technique of shoot-tip culture (see Chapter 8 , this vol.) may be more expedient for plant propagation. In this procedure, the explant is still a dissected shoot apex, but a much larger one, containing a relatively large number of developing leaf primordia. Typically, the explant is between 3-4 mm and 2 cm in length, and development in vitro is still regulated so as to be confined to outgrowth of an organized shoot, without adventitious propagation.

Freeze-etch observations of protoplasts isolated from tobacco (Nicotiana tabacum L.) mesophyll tissue and tomato (Lycopersicum esculentum Mill.) fruit locule tissue are described which clarify earlier observations (Burgess, J., Fleming, E.N., Planta 131, 173-178, 1976; Planta 133, 267-273, 1977), obtained using scanning electron microscopy. of "fibres" associated with "projections" from these cell surfaces. It is demonstrated (1) that the "fibres" consist of bundles of small numbers of microfibrils which have become artifactually thickened by the deposition of coating materials, and (2) that the apparent association between "fibres" and "projections" results from microfibrils being lifted preferentially from protoplast surfaces in regions rich in "projections" (plasmalemmasomes). With the higher resolution available using freeze-etching it can be demonstrated that microfibril deposition does not occur in discontinuous zones on these protoplast surfaces. Globules associated with microfibril termini in radish (Raphanus sativus L.) roots are illustrated and it is proposed that turgor pressure differences between isolated protoplasts and intact tissue may account for the absence of similar globules from isolated protoplast surfaces.

The reappearance of cellulose microfibrils at the naked surface of protoplasts enzymatically isolated from tobacco (Nicotiana tabacum L. var. Xanthi) mesophyll tissue has been closely studied using the techniques of thin-sectoining and the deep-etch modification of the freeze fracture procedure.A 16 h lag period was recorded between the time of isolation and the sudden appearance of considerable lengths of cellulose microfibril at the outer protoplast surface. The microfibrils were not associated with any structured particles or apparently differentiated regions of the plasmalemma. Terminal regions of the microfibrils appeared to have tapering ends, or else be sinking into the membrane substance. There was no evidence to suggest transport of intact microfibrils in vesicles through the cytoplasm to the plasmalemma.The reported observations have been discussed with respect to the various working hypotheses which have been proposed for the 'in vivo' construction of cellulose microfibrils.