Moreover, protocorm-like bodies of Dendrobium orchid and Citrus madurensis embryonic axes required shorter PVS exposure times at 25uC than 0uC, i.e. 20 versus 60 min respectively. Whilst the time window for optimum PVS treatment is wider at lower temperatures, tropical species tend to respond better with warmer temperature treatment, e.g. Colocasia esculenta. The physical dimensions and permeability characteristics of the tissue under investigation profoundly affect the outcome of the cryoprotection and cryopreservation procedures. Smaller apices of garlic displayed higher regeneration after cryopreservation than large ones. Similarly, Nephelium ramboutan-ake shoot-tips of c. 2 mm tolerated cryopreservation well, as did 0.8 mm diameter axillary buds of Colocasia esculenta. The cryopreservation of mature zygotic embryos of recalcitrant seeds generally requires a reduction in tissue mass to facilitate cryoprotectant uptake. Usually, this involves the excision of the embryonic axis. In axes of recalcitrant seeds of sweet chestnut such surgical intervention results in a burst of superoxide, with further oxidative stress during subsequent desiccation. In this context, assuming that the protectants permeate sufficiently. Permeation of chemicals into the intercellular spaces and cells of plant tissues is compounded by many features. To enable the rapid permeation of the viability stain, triphenyl tetrazolium chloride, into oily tissues of pine seed, we previously used vacuum infiltration. Similarly, this system has been used to improve efficient gene transformation, the delivery of pathogenic bacteria into the intercellular spaces of plants to study pathogenplant cell interactions and the diffusion of an inhibitor of ethylene action so that pear fruits have prolonged storage. In addition, preliminary studies have shown that vacuum-assisted glycerol cryoprotectant infiltration can preserve the normal histology of rat leg muscle with no ice crystal formation after 3 weeks storage at 280uC. In this study, we developed and compared the efficacy of vacuum infiltration vitrification using PVS2 for the cryopreservation of seed embryos of three species with varying morphology, stress physiology and chemistry: Carica papaya ; Passiflora edulis ; and Laurus nobilis. These species have purported differences in seed storage characteristics. C. papaya has a high level of desiccation tolerance to about 5% moisture content, limited storability at 220uC, but tolerance of cryopreservation. P. edulis seeds may show reduced viability after drying to 5–6% moisture content, but the majority of dry seeds tolerate cryopreservation. Both species have spatulate embryos in copious endosperm. Finally, L. nobilis has seeds with a lowest safe moisture content of c. 24%, below which they are desiccation sensitive and successful moist storage at 0uC is limited to about 4 months.