Stress and cell cycle regulation during somatic embryogenesis plays a key role in oil palm callus development.

Oil palm is an oleaginous plant of relevant economic importance since its fruits are rich in vegetable oil. These plants have a single apical meristem and the main method for vegetative propagation is somatic embryogenesis. The aim of this study was to identify differentially abundant proteins from oil palm genotypes contrasting in the capacity of embryogenic competence acquisition, using shotgun proteomics. Oil palm leaves were subjected to callus induction and the material was collected in biological triplicates at 14 and 90 days of callus induction. LC-MS/MS analysis was performed and revealed a total of 4695 proteins. Responsive and non-responsive genotypes were compared at 14 and 90 days of callus induction and 221 differentially abundant proteins were obtained. The data analysis revealed several proteins mainly related to energy metabolism, stress response and regulation of cell cycle, further analyzed by qRT-PCR, which seem important for embryogenic development. We suggest some of these proteins as key factors for the success of callus formation in oil palm including antioxidant and cell division proteins as well as proteins involved in the ubiquitination pathway. These proteins may also be potential biomarkers for the acquisition of embryogenic competence. SIGNIFICANCE: Antioxidant and cell division proteins as well as proteins involved in the ubiquitination pathway are key factors for the success of callus formation in oil palm. The proteins identified in this study may be potential biomarkers for embryogenic competence acquisition.

Genotype-dependent changes of gene expression during somatic embryogenesis in oil palm hybrids (Elaeis oleifera x E. guineensis).

To understand the molecular processes triggered during the different steps of somatic embryogenesis (SE) in oil palm, the expression of 19 genes associated to SE identified in proteomic and transcriptomic studies was investigated by qRT-PCR. To evaluate the differential expression of these genes, two interspecific hybrid genotypes (Elaeis oleifera x Elaeis guineensis) contrasting for the acquisition of embryogenic competence were used. Aclorophyllated leaves of both hybrids, one responsive (B351733) and the other non-responsive (B352933) to SE were submitted to callus induction and collected at different time points: 0 (before induction), 14, 30, 90 and 150 days of callus induction (doi). The results obtained showed that all evaluated genes were downregulated at 14 doi in the responsive genotype when compared to the non-responsive. It was also possible to observe that most of the genes changed their expression behavior at 30 doi and were upregulated thereafter until 150 doi, with the exception of the pathogenesis-related PRB1-3-like (PRB1-3) gene, which did not show differential expression at 30 doi and was downregulated at 90 and 150 doi when compared to the non-responsive hybrid. These results indicate that 30 doi is a turning point in gene expression, probably associated to embryogenic competence acquisition. We also show that the expression behavior of the responsive genotype is more stable than that of the non-responsive when the different induction time points are compared to 0 doi (before induction). Moreover, the results obtained in this study corroborate our hypothesis that the regulation of genes involved in the control of oxidative stress and energy metabolism are crucial for the acquisition of embryogenic competence in oil palm.

In vitro rescue of interspecific embryos from Elaeis guineensis x E. oleifera (Arecaceae).

The African oil palm (Elaeis guineensis) is the most effective oil producer in tons per hectare. Nevertheless, its increasing cultivation in Latin America is harmed by the "lethal yellowing". Genetic resistance to this anomaly can be found in the germplasm of American oil palm or caiaué (E. oleifera), a native species from the Amazon rainforest. However, the procedures adopted to induce seeds of E. guineensis to germination frequently result mild for interespecific hybrids. Embryo in vitro cultivation can be a viable option. This work was aimed initially to test liquid MS medium supplemented with different glucose or sucrose concentrations for the in vitro cultivation of zygotic embryos from E. guineensis x E. oleifera controlled pollinations. Additionally we investigated different compost mixtures to acclimatize the regenerated hybrid plantlets. Concentrations of 10, 20 and 30g/L of both sugars were tested on flasks containing five mature zygotic embryos, with 15 repetitions per treatment in a total of 450 explants. The number of embryos displaying shoots and radicles at least 2mm in length per experimental unit was evaluated during phase one of in vitro cultivation. Plantlets displaying shoots and radicles were transferred to phase two of in vitro cultivation and subsequently to acclimatization, under 70% shading with manual water supply. The experiments of acclimatization were conducted with 130 plantlets randomly distributed in pure horticultural compost, 3:1 or 1:1 compost:sand mixtures and each plantlet was defined as an experimental unit. Data were submitted to ANOVA, t test and analyzes of correlation (p < or = 0.05). Highest emergence rates were 97% for shoots and 73% for radicles, observed in MS medium supplemented with 20g/L (110mM) of glucose. This sugar in concentrations of 20 or 30g/L provided balanced shoot/root development, and this was considered one of the reasons for the higher frequency of plantlet establishment. The survival percentage was 55% after the first 43 days of acclimatization and by the fourth month, 66 plants developed simultaneously longer shoot and root systems in pure horticultural compost. In conclusion, radicle development was an impairment to plantlet establishment and was overcame under media with glucose above 110mM. Acclimatization could benefit from an extended period of in vitro development.