Sarah Ashmore

Assessment of genetic and epigenetic changes following cryopreservation in papaya

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.

Genetic mapping of a PRSV-P resistance gene in "highland papaya" based on inheritance of RAF markers

Papaya ringspot virus type P (PRSV-P) is a significant disease of Carica papaya. A major gene for PRSV-P resistance has been mapped in Vasconcellea cundinamarcensis, a distant relative of C. papaya. This was achieved by genetic mapping of the resistance phenotype and inherited, dominant, polymorphic randomly amplified DNA fingerprint (RAF) markers in F2 progenies of V. parviflora and V. cundinamarcensis. The parents of this cross confer resistance to several major diseases that affect C. papaya including PRSV-P in V. cundinamarcensis. Heredity of DNA markers and PRSV-P resistance was studied in the intrageneric population presented due to intergeneric fertility barriers between Carica and Vasconcellea. Genetic polymorphism between parents, based on RAF markers, was 75% with more than 70% of markers generated showing mendelian segregation for the expected ratios 1:3 or 1:1 (p < 0.05). Preferential inheritance of markers from either parent was not detected in the F2, indicating stable transfer of the genetic material. Discrete V. parviflora and V. cundinamarcensis linkage maps were compiled from 79 and 83 framework markers, delineating to 10 and 11 groups respectively. F1 and F2 progeny were screened for resistance to PRSV-P under controlled conditions. The resistant phenotype segregated 3:1 in the F2 and mapped to V. cundinamarcensis linkage group 7 with adjacent RAF markers within 4 cM. The framework maps of V. parviflora and V. cundinamarcensis presented cover 630.2 and 745.4 cM respectively, accounting for between 47–52 and 49–55 percent of the predicted genome lengths. These maps provide a platform for further genetic study of disease resistance characteristics identified in these species and the development of DNA markers tightly linked to these traits, which could be applied to the breeding of resistant C. papaya cultivars.

Conservation technologies for safeguarding and restoring threatened flora: case studies from Eastern Australia

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.

Cytogenetic studies of Haplopappus gracilis in both callus and suspension cell cultures

SummaryInvestigations have been carried out on karyotype change in both callus and suspension cell cultures of Haplopappus gracilis (2n=4). It has been found that polyploidization arises directly in culture to give up to six times the normal diploid chromosome number in some cultures. In polyploid cultures, both chromosome loss and chromosome rearrangements occur to give rise to aneuploid karyotypes displaying chromosomes which differ in morphology from the diploid set. Whole or partial chromosome loss has been observed in the form of lagging chromosomes and chromosome bridges at anaphase, and micronuclei, ring chromosomes and chromosome fragments at other stages in mitosis. C-banded preparations have confirmed the occurrence of chromosomal rearrangements. Comparative investigations suggest that (i) more polyploidy occurs in callus cultures than in suspension cell cultures, and (ii) the presence of cytokinin (kinetin) in the culture medium may reduce the extent of karyotype change.

Field performance evaluation and genetic integrity assessment of cryopreserved papaya clones

This paper is the first report of field performance and evaluation of morphological traits following cryopreservation in four genotypes of Carica papaya (Z6, 97, TS2 and 35). It also describes the successful establishment of in vitro plantlets following vitrification-based cryopreservation of shoot tips and their acclimatisation through to field establishment. Cloned plants resulting from untreated controls, as well as controls taken at three other stages of the cryopreservation process (dissection, pre-treatment, plant vitrification solution 2 (PVS2) treatment) and cryopreserved plants were established to ensure a rigorous appraisal of any variation. Results indicate no differences between any of the control plants or cryopreserved plants for either growth performance or morphology. In addition, both randomly amplified DNA fingerprinting and amplified DNA methylation polymorphism markers were used to assess any genomic or methylation changes in genotype 97 at four different developmental stages post cryopreservation (in vitro, acclimatisation and field). Only small genomic DNA modifications (0–8.3%) were detected in field stage plants and methylation modifications (0–4.3%) were detected at both the in vitro and field stages for samples treated with PVS2 or cryopreservation.

Interaction of purified DNA with plant protoplasts of different cell cycle stage: The concept of a competent phase for plant cell transformation

SummaryThe polycation mediated attachment of purified tritiated DNA to plant protoplasts has been measured by quantitative microautoradiography. The automated grain counting technique used, also provides information on the cell cycle stage of individual protoplasts, which circumvents the need to synchronize the plant cell population before preparation of protoplasts. With protoplasts from asynchronously dividing suspension cultures of Nicotiana syhestris (NS-1), S-phase protoplasts appear to be inefficient binders of 3H-DNA, as compared with G1 or G2 protoplasts. Protoplasts derived from a tumour line of Crepis capillaris (CAPT) exhibit 3H-DNA binding at all cell cycle phases, but Sphase protoplasts appear to be preferential binders. These differences are discussed with reference to cell cycle kinetics, membrane charge variation and the possibility of increasing the efficiency of genetic transformation of higher plant cells in culture.

Vitrification-based shoot tip cryopreservation of Carica papaya and a wild relative Vasconcellea pubescens

This paper reports on the effects of pre- and post-liquid nitrogen modifications to a previously published protocol for vitrification-based cryopreservation of papaya (Carica papaya L.) shoot tips. The aim was to improve the protocol for application across a wider range of papaya genotypes. Results showed that recovery from cryopreservation is genotype dependent, but the post-subculture age of the shoot tips was not significant in the two genotypes tested. Pre-culture for 2 days gave greater recovery than that for 0, 1 or 4 days. The duration and temperature of exposure to plant vitrification solution 2 (PVS2) had the most significant impact, with optimal recovery of 60 and 64% with 10 min at room temperature or 20 min at 0°C, respectively. Exposure to PVS2 for greater than 30 min reduced recovery to below 20%. Post-cryopreservation recovery was highest in media containing 1.0 µm 6-benzylaminopurine (BAP) or a combination of BAP and gibberellic acid (GA3) (1.0 and 0.5 µm, respectively). Incubation in the dark for the first 24–48 h had no significant effect on recovery. A refined protocol was developed based on these results and application of this protocol proved to be effective across seven papaya genotypes and one related species, Vasconcellea pubescens V.M.Badillo. Genotypes previously showing no survival on an unrefined protocol recovered using this revised protocol.

Seed morphology and ultrastructure in Citrus garrawayi (Rutaceae) in relation to germinability

Combinational traits of seed size and seed-coat hardness in Citrus garrawayi (F.M.Bailey) (syn. of Microcitrus garrowayi) were investigated as markers for estimation of seed morphological and physiological maturity. Seed size (length) and coat hardness correlated well with changes in seed coat and embryo morphological development, dry-weight accumulation, decreases in moisture content and a significant increase in germinability. Seed moisture content decreased from 82 ± 1% in immature seeds to 40 ± 1% at seed maturation. The outer integument of immature seeds consisted of thin-walled epidermal fibres from which outgrowths of emerging protrusions were observed. In comparison, mature seed coats were characterised by the thickening of the cell walls of the epidermal fibres from which arose numerous protrusions covered by an extensive mucilage layer. Immature seeds, with incomplete embryo and seed-coat histodiffereniation, had a low mean germination percentage of 4 ± 4%. Premature seeds, with a differentiated embryonic axis, were capable of much higher levels of germination (51 ± 10%) before the attainment of mass maturity. Mature seeds, with the most well differentiated embryonic axis and maximum mean dry weight, had the significantly highest level of germination (88 ± 3%).

Micropropagation of Two Australian Native Fruit Species, Davidsonia pruriens and Davidsonia jerseyana G. Harden & J.B. Williams

Nodal explants from in vitro grown seedlings of Davidsonia pruriens and D. jerseyana, established on MS media were treated with various concentrations of three cytokinins. D. pruriens developed optimum shoot growth in terms of shoot height and number of leaves per shoot when 1.0 µM BA was added to basal MS medium while optimum shoot growth for D. jerseyana was obtained when 0.01 µM 2iP was added to the medium. Optimum root initiation and development was obtained when actively growing axillary shoots were cultured on 1/2MS medium plus 32.2 µM IBA for 3–5 days for D. pruriens and 2–3 days for D. jerseyana before transfer to PGR-free medium containing 10 µM riboflavin. Root initiation of more than 80% was achieved with multiple genotypes of D. pruriens and three genotypes of D. jerseyana using juvenile material. The plantlets were transferred to pots and grown in the greenhouse with a success rate of 60% for D. pruriens and 75% for D. jerseyana. Adult D. jerseyana stem explants produced 2–5 shoots per nodal explant upon treatment with 0.1 µM BA. Side shoots from adult D. jerseyana produced similar results for shoot multiplication as for juvenile material. Protocol for multiplication of adult D. pruriens was achieved with much greater difficulty by using material from the green house. Axillary shoots were initiated when 100 µM TDZ was applied to the stem of an adult pot plant and the resultant side shoots were cultured on MS medium containing 1.0 µM BA and 1.0 µM GA3.

Morphological characterization of seeds of three Australian wild Citrus species (Rutaceae): Citrus australasica F. Muell., C. inodora F.M. Bailey and C. garrawayi F.M. Bailey

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.