The ultimate aim of this study was the generation of haploid embryos from oil palm (Elaeis guineensis Jacq.) microspores. A range of factors leading to the induction and regeneration of haploids via anther culture has been investigated.
The timing of microspore development from the microspore mother cell through uninucleate and binucleate stages to mature pollen has been determined. Late uninucleate to early binucleate microspores, which are optimal for androgenesis, occur predominantly in male inflorescences from the leaf axils of fronds (leaves) 16 and 17. A safe and effective decontamination technique has been developed which involves the partial dissection of the floret and adjustment of the pH of the NaOCI (0.5%) to pH 5-6. In vitro techniques were used to investigate haploid induction and regeneration.
Oil palm anthers incubated at a low temperature of 5°C for 96 hours or treated with 0.4M mannitol for 24 hours produced multicellular structures from microspores. While high temperature (30, 35 or 40°C) induced extensive morphological changes in microspores, it did not lead to androgenesis. Low temperature was adopted as the standard protocol to avoid any possible interference by mannitol in the biochemical or biophysical pathways leading to androgenesis.
The greatest number of multicellular structures was obtained when six anthers were cultured per 2ml of medium. MCB medium containing 250M maltose and 25mM inorganic nitrogen (N03:NH4 ratio of 80:20) produced higher numbers of multicellular structures than did oil palm specific callogenic (U2) or embryogenic (E97 A) media. Anthers produced significantly higher numbers of multicellular structures when cultured under some form of continuous light. Microspore responses to white (0.85%), blue (0.72%) or red (0.57%) wavelengths were significantly greater than from continuous darkness (0.36%). MCB medium was not enhanced by the addition of NAA (0.1, 1.0 or 10mg/l). While the production of multicellular structures varied between individual palms (0-7.9%), no significant genotypic effect was found within any treatment.
Low temperature stress was accompanied by a change in the DNA and protein patterns detected. Radio labelling (33P) of the genomic and Hpa II digested DNA revealed changes associated with androgenesis. No changes in DNA methylation patterns were detected using the three-oligonucleotide primers AJ-15, AT-10 and BB- 18. However deletions and/or insertions in the DNA sequence were observed. Additional protein bands were detected at 15, 18 and 27 kDa. The presence of these additional bands was increased with increasing anther size. These changes in the DNA and protein banding patterns clearly show a change at the molecular level in response to the low temperature induction treatment.
This thesis provides the first clear evidence of the initiation of androgenesis from oil palm microspores. Multicellular structures were obtained from microspores in response to low temperature induction. A coincidental change in DNA and protein banding patterns was recorded. It remains to demonstrate a causal link between these two phenomena. The challenge also remains to regenerate plants from these microspore derived multicellular structures.