Roderick Alastair Drew

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 diversity revealed in the apomictic fruit species Garcinia mangostana L. (mangosteen)

The novel molecular marker technique Randomly Amplified DNA Fingerprinting (RAF)was used to survey genetic relationships between 37 accessions of the tropical fruit G. mangostana (mangosteen) and among 11 accessions from eight other Garcinia species. Although mangosteen is believed to reproduce exclusively through apomixis, our results show that considerable genetic diversity exists within G. mangostana and between other Garcinia species. Among the 37G. mangostana accessions examined, nine different genotypes were identified which clustered into three distinct groups based on correspondence analysis(reciprocal averaging). For 26 (70%) of the accessions no marker variation was detected over 530 loci screened. A further eight (22%) accessions exhibited very low levels of variation (0.2–1%) suggesting at least one well conserved mangosteen genotype. The remaining three accessions (8%) showed extensive variation (22–31%)compared with the majority of accessions. The three mangosteen groups were 63–70% dissimilar to the other Garciniaspecies investigated. The genetic diversity identified in this research will assist in the conservation of Garciniagermplasm and provides a valuable framework for the genetic improvement of mangosteen.

Genetically engineered immunity to Papaya ringspot virus in Australian papaya cultivars

Papaya ringspot virus (PRSV), which has recently been identified inAustralia, is the major limiting factor in papaya production worldwide. In thispaper we report the development of two Australian papaya cultivars that areimmune to infection with PRSV. Papaya somatic embryos were transformed viamicroprojectile bombardment using a construct containing an untranslatable PRSVcoat protein coding region. Immunity was demonstrated by repeated inoculationinthe glasshouse and eighteen months in the field. The immune lines were shown tohave up to five copies of the transgene by Southern hybridisation. Northernhybridisation showed that the coat protein transcript in the immune linesappeared to be degraded; therefore, the mechanism of resistance appears to bepost transcriptional gene silencing via RNA degradation. We have taken aproactive approach to control of PRSV in Australia by developing geneticallyengineered resistance before the Australian papaya industry has been decimatedby the inevitable spread of PRSV.

Morphological, molecular and cytological analyses of Carica papaya x C. cauliflora interspecific hybrids

Abstract Morphological, molecular and cytological analyses were performed to assess the hybridity of 120 putative interspecific hybrids of Carica papaya L.×C. cauliflora Jacq. In the putative interspecific hybrids the number of main leaf veins was intermediate between the two parents while the hermaphrodite flower sex form and the low vigour were distinctive features of these hybrids. Petiole length, stem diameter, leaf length, leaf width and flower colour were similar to C. papaya, whereas leaf shape, type, serration, venation, petiole hairiness and flower shape were similar to C. cauliflora. Markers generated by the polymerase chain reaction using 72 10-mer primers (random amplified polymorphic DNA) revealed a high level of polymorphism (64%) between C. papaya and C. cauliflora. Seventeen of these primers yielded reliable and easily scorable polymorphic banding patterns that were further screened to reveal hybrids. A range of 1–5 RAPD primers consistently confirmed that all 120 plants were genetic hybrids, with all of them containing at least one band from the male parent. Cytological analysis revealed that 7–48% of the cells in many of the interspecific hybrids were aneuploid suggesting that chromosome elimination was occurring. The frequency of aneuploid cells was negatively associated (r=0.88) with the number of bands from the male parent integrated into the hybrid. Pollen fertility of the hybrids was from 0.5 to 14.0% while C. papaya and C. cauliflora had 88.0–99.0% and 90.0–97.0% fertile pollen, respectively.

In vitro culture of adult and juvenile bud explants of Passiflora species

Cultivar E23, an F1 hybrid of P. edulis and P. edulis f. flavicarpa is usually propagated by shoot-tip grafting. Various media were tested to evaluate the potential of E23 for in vitro propagation. Adult tissue was difficult to culture and did not respond to media containing low (<10 µM) concentrations of growth regulators. Growth of adult buds on intact stem sections was promoted by 1 week of dark incubation on MS basal medium plus 150 µM 2iP, 200 µM adenine sulphate and 17.1 µM IAA (3 mg l−1), and further developed into shoots on MS medium plus 4.9 µM 2iP (1 mg l−1) and 5.7 µM IAA (1 mg l−1). By contrast, juvenile shoots of E23, and Passiflora species: edulis f. flavicarpa, edulis, alata, caerulea, mollissima, coccinea, herbertiana and suberosa grew rapidly on MS medium plus 10 µM kinetin and 5 µM IAA. Rapid multiplication was achieved on MS plus 20 µM BA, 10 µM kinetin, 5 µM IAA, and roots initiated on MS plus 5 µM IAA.

Degradation of exogenous indole-3-butyric acid and riboflavin and their influence on rooting response of papaya in vitro

Varying concentrations of riboflavin were added to a De Fossard et al. (1974) basal medium containing 10 µM IBA and the effect on adventitious root initiation on shoots of Carica papaya L. was studied. Ninety percent root initiation occurred in 11 days when 1 µM riboflavin was added to the culture medium. Smaller rooting percentages were observed and roots emerged more slowly with riboflavin concentrations greater and less than 1 µM. Tissue culture media were maintained at 27°±1°C in either darkness or 12-h photoperiods for 28 days, and concentrations of riboflavin and IBA were measured at regular intervals using HPLC analysis. In a De Fossard et al. (1974) basal medium, riboflavin concentrations (0.1, 1.0, 10.0 µM) decreased rapidly in light and were independent of the presence of IBA. IBA concentration steadily decreased when media was placed in light, and increasing riboflavin concentrations accelerated the reduction of IBA levels. Concentrations of IBA and riboflavin were stable with dark incubation.

Effect of ethylene and culture environment on development of papaya nodal cultures

Papaya (Carica papaya L.) nodal cultures modified the atmosphere of the headspace of the vessel used for culture maintenance by producing ethylene. Under culture maintenance nodal cultures grew poorly and leaves senesced. Incubating nodal cultures under a range of ethylene concentrations suggested that this poor performance was caused in part, by the production of ethylene and its accumulation in the headspace of the vessel. To further evaluate the role of ethylene accumulation in growth suppression, aminoethoxyvinylglycine (AVG), 1-aminocyclopropane-1-carboxylic acid (ACC) and silver thiosulphate (STS), were added to the nutrient medium and ethylene measurements performed during culture growth. The ethylene-suppressant, AVG, (1.2 µM) and the ethylene-antagonist, STS, (0.3 mM) significantly improved nodal culture growth (283 and 289% respectively), leaf area production (350 and 211% respectively) and reduced leaf senescence, while the ethylene-precursor, ACC, (1.5 mM) significantly decreased culture growth (71%), leaf area production (88%) and promoted leaf senescence. Furthermore, nodal culture growth was significantly better at 20 °C than 30 °C since ethylene production and accumulation were less in these conditions. Better control or management of ethylene accumulation produces healthier nodal cultures for micro-propagation and may be a way of improving productivity of other papaya shoot culture systems.

An Improved Embryo-Rescue Protocol for a Carica Interspecific Hybrid

An improved embryo-rescue protocol was developed for embryos (90 days old) of Carica papaya L. (Clone 2001), and subsequently was utilised for efficient production of interspecific hybrids of C. papaya × C. cauliflora Jacq. from 90- to 120-day-old embryos. Pre-incubation of C. papaya embryos for 7 days on a germination medium containing half-strength De Fossard nutrients supplemented with gibberellic acid (10 μM), 6-benzylamino-purine (0.25 μM), alpha-naphthalene-acetic acid (0.25 μM). sucrose (58 mM) and agar (8 g L-1) supported 100% germination. Subsequent transfer of germinated embryos to a nutrient medium that was identical, except that it was free of plant growth regulator, allowed good growth but induced shoot etiolation and callus production. Reducing the pre-incubation of C. papaya embryos on this medium to 5 days before transfer to the medium free of plant growth regulator produced similarly high germination (96%), but allowed for the production of good quality seedlings that were unetiolated and free of unwanted callus. For interspecific hybrids, a 5-day pre-incubation of the embryos on a liquid formulation was better than the solid formulation as it promoted better growth and vigour of the normally abortive interspecific hybrid embryos. Using the improved protocol, 1981 of 2100 (94%) interspecific hybrid embryos consisting of single and multiple forms were germinated. In all cases, the germinating multiple embryos underwent further embryogenesis that allowed for the production of 485 (25%) morphologically normal hybrid plants grown in soil in the glasshouse.

Rhizogenesis and root growth of Carica papaya L.in vitro in relation to auxin sensitive phases and use of riboflavin

High rooting percentages and high-quality adventitious root systems for papaya (Carica papaya L.) were obtainedin vitro by appropriate auxin source, duration of exposure to auxin and use of riboflavin. Root initiation of papaya shoots was higher using IBA than IAA, NAA or PCPA. Maximum rooting percentage (96%) was achieved by exposure of shoots to a medium containing 10 µM IBA for 3 days before transfer to a hormone-free medium. However, the resultant plants had small shoots and callused roots. Shoot and root growth were improved when shoots were transferred after 2 days from medium containing 10 µM IBA to hormone-free medium containing 10 µM riboflavin. Good root initiation, and root and shoot growth were also obtained when shoots were incubated for 2 days in darkness on a medium containing 10 µM IBA and 31 µM riboflavin before transfer to light. Alternatively, cultures could be placed in the light on medium containing 10 µM IBA, and after 1 day the medium overlaid with 300 µM riboflavin (1 ml over 10 ml of medium).

Efficient organogenesis of an Australian passionfruit hybrid (Passiflora edulis x Passiflora edulis var. flavicarpa) suitable for gene delivery

An efficient regeneration protocol based on organogenesis from cotyledon explants and suitable for gene delivery has been developed for an Australian passionfruit hybrid. Multiple shoots were regenerated from 30-day-old cotyledon explants on Murashige and Skoog (MS) medium containing 6-benzylvaminopurine (BAP) and coconut water. Media pulsing experiments were conducted to investigate the effect on organogenesis of exposure time of the explants to MS containing 10 mu M BAP and 10% (v/v) coconut water, i.e. passionfruit regeneration medium (PRM). Continuous exposure of these explants to PRM maximised the number of shoots produced to 12.1 per explant. However, periods on hormone-free medium improved the appearance of the shoots and increased the number of explants with shoots from 75 to 84.6%. Further, shoots exposed for 7 days to half-strength MS supplemented with 10 mu M NAA (1-napthalene acetic acid) produced twice as many plantlets than those on half-strength MS alone. Transient GUS histochemical assays indicated delivery of the uidA gene via Agrobacterium tumefaciens.