P. M. Magdalita

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.

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.

Application of Biotechnology to Carica Papaya and Related Species

An efficient protocol has been developed for clonal multiplication of papaya based on rooted microcuttings from nodes of apically dominant plants in vitro. Other Carica species cauliflora, parviflora, pubescens and goudotiana have been micropropagated using this system. Four thousand tissue-cultured plants of one papaya clone have been established in the field with no apparent off-types. Plants established from adult tissue exhibited reduced juvenility compared with seedlings. Immature embryos of crosses between C. papaya and C. cauliflora have been rescued and grown in vitro. Three hundred and fifty interspecific hybrids have been established in pots and distinguished from parents by morphology and RAPD markers. Hybrids are being screened for resistance to papaya ringspot virus type P (PRSV-P) by sap inoculation followed by ELISA serological assay. Techniques are being developed for production of transgenic papaya plants resistant to PRSV-P. Transformation systems being investigated are microprojectile bombardment and the use of Agrobacterium tumefaciens. Plantlets are being regenerated via embryogenesis from immature embryos rescued from seeds, and from somatic embryos in vitro.