A vitrification based cryopreservation technique for storage of in vitro shoot tips of papaya has been tested to ensure applicability across a range of genotypes and to assess the stability of both genotype and phenotype of such clonal material following cryopreservation. Shoot tips of 12 genotypes were cryopreserved, recovery rates were determined and resultant plants were screened for genetic and epigenetic changes. Genomic DNA structure was explored using polymerase chain reaction (PCR) based randomly amplified DNA fingerprinting (RAF), and methylation patterns were monitored using the amplified DNA methylation polymorphism (AMP) PCR technique. Plantlets were recovered following cryopreservation in all but one genotype and recovery rates of 61-73% were obtained from six genotypes. The regenerated plantlets showed varying levels of genomic DNA modifications (0-10.07%), and methylation modifications (0.52-6.62%) of detected markers. These findings have not been reported previously for papaya, and indicate some genotype dependent variability in DNA modifications occur following cryopreservation which may result in somaclonal variation.

This paper highlights recent advances and improved scientific understanding of conservation technologies through selected case studies on threatened plant species indigenous to Eastern Australia. This includes investigations into seed desiccation, storage responses and cryopreservation in rainforest species, particularly the socio-economically important Australian native Citrus spp., Davidsonia spp. (Davidson's plum) and Syzygium spp. This work also (1) increases our understanding of ecological correlates of seed desiccation sensitivity for predictive use and (2) improves restoration practice through better understanding of seed storage and germination requirements. The use of in vitro conservation technologies in support of conservation actions for endangered species is outlined in case studies on Wollemia nobilis (Wollemi pine), epiphytic and terrestrial orchid species, and an endangered fern species.

The comparative morphology of the seeds of three Australian Citrus species, C. australasica C. inodora and C. garrawayi, was studied. Their seed characteristics were broadly similar to those of the cultivated species of the genus, when observed under light and scanning electron microscopy. Citrus garrawayi differed in seed shape (rounded to triangular) and seed coat morphology (i.e., thicker with longer epidermal protrusions) from C. australasica and C. inodora (rounded surface with flat underside in shape). The well-developed minute epidermal protrusions on the seed coat of C. garrawayi were more similar to those in the cultivated species, C. · sinensis and C. · aurantium. In contrast, the surface topography of C. australasica and C. inodora seeds was more like that of the cultivated species, C. · aurantifolia and C. · limon. Seed morphology, especially surface topography, was found to be a useful tool for taxonomic identification in Australian wild citrus.

Development of resistant papaya varieties is widely considered the best strategy for long-term control of the papaya ringspot virus type P (PRSV-P). Several species of "highland papaya" from the related genus Vasconcellea exhibit complete resistance to PRSV-P, and present a valuable source of resistance genes with potential for application in Carica papaya. The objectives of this study were two fold; to identify molecular markers linked to a previously characterised PRSV-P resistance gene in V. cundinamarcensis (psrv-1), and to develop codominant marker based strategies for reliable selection of PRSV-P resistant genotypes. Using a bulked segregant analysis approach, dominant randomly amplified DNA fingerprint (RAF) markers linked to prsv-1 were revealed in the resistant DNA bulk, which comprised F2 progeny from a V. parviflora (susceptible) x V. cundinamarcensis (resistant) interspecific cross. One marker, Opk4_1r, mapped adjacent to the prsv-1 locus at 5.4 cM, while a second, Opa11_5r, collocated with it. Sequence characterisation of the Opk4_1r marker permitted its conversion into a codominant CAPS marker (PsiIk4), diagnostic for the resistant genotype based on digestion with the restriction endonuclease PsiI. This marker mapped within 2 cM of the prsv-1 locus. Psilk4 was shown to correctly identify resistant genotypes 99% of the time when applied to interspecific F2 progeny segregating for the resistant character, and has potential for application in breeding programs aimed to deliver the PRSV-P resistance gene from V. cundinamarcensis into C. papaya.

Shoot tips and seeds of papaya (Carica papaya L.) were successfully cryopreserved. Shoot tips were incubated for 1 to 6 days before vitrification and optimum treatment time was 1-4 days. Duration of exposure to vitrification solution was varied and 70% recovery was obtained from the shoot tips which had been exposed to 100% PVS2 for 20 minutes at 0 o C. Treatments for less than 20 minutes or more than 40 minutes resulted in no regeneration after liquid nitrogen (LN) treatment. Shoot tips closer to the apex had almost a twofold rate of recovery compared with tips excised from the basal part of the shoot. Seeds of a southeast Queensland genotype were desiccated to moisture contents of 40%, 20%, 15%, 10%, and 5% (wet weight basis), followed by freezing in liquid nitrogen (LN). Germination rate of seeds that had been desiccated but not cryopreserved were consistently high (between 70% and 90%) across the range of moisture contents down to 5%. For seeds frozen in LN, the germination rate of seeds at 100%, 40%, 20% and 15% moisture content was 0%, 8%, 6%, and 8% respectively. A substantial increase in germination rate to 48% was achieved at 10% moisture content, declining to 20% at 5% moisture content for cryopreserved seeds. Within 10 weeks of transplanting to the field, growth rates of plants re-grown from cryopreserved seeds were significantly reduced (25%) when compared with the control plants. Substantially retarded growth was observed in one plant re-grown from a cryopreserved shoot tip, after 2.5 years in the field.