Aperfeiçoamento da embriogênese somática, reprodução e análise molecular de retrocruzamentos interespecíficos em palma de óleo (Elaeis spp.)

Abstract

In this work, three Chapters are proposed: 1) Optimization of somatic embryogenesis in oil palm (Elaeis spp.) from mature zygotic embryos; 2) Influence of initial cutting thickness on somatic embryos differentiation in oil palm (Elaeis spp.) using the thin cell layer technique (TCL), and; 3) Vegetative reproduction and molecular analysis of interspecific backcrosses of oil palm regenerated by somatic embryogenesis. In the first Chapter, we developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from mature zygotic embryos of oil palm. Embryogenic calli were induced from mature zygotic embryos of oil palm on modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid or picloram, alone or in combination with activated charcoal. The greatest frequency of embryogenic callus induction (97.5%) was obtained by culturing mature zygotic embryos on callus induction medium with 450 μM picloram and 2.5 gL−1 activated charcoal. Embryogenic calli proliferated on a medium with a reduced concentration of picloram. Embryogenic calli were then subcultured on a medium supplemented with 12.3 μM 2-isopentenyladenine and 0.54 μM naphthaleneacetic acid, with subcultures at 4-wk intervals. Somatic embryos were regenerated on a medium with Murashige and Skoog macro- and micronutrients at halfstrength concentrations supplemented with 20 g.L−1 sucrose, 2.5 g.L−1 activated charcoal, and 2.5 g.L−1 Phytagel. Detailed histological analysis revealed that somatic embryogenesis followed an indirect pathway. Primary calli were observed after 4–6 wk of culture and progressed to embryogenic calli at 12 wk. Embryogenic cells exhibited dense protoplasm, a high nucleoplasmic ratio, and small starch grains. Proembryos, which seemed to have a multicellular origin, formed after 16–20 wk of culture and successive cell divisions. Differentiated somatic embryos had a haustorium, a plumule, and the first and second foliar sheaths. In differentiated embryos, the radicular protrusion was not apparent because it generally does not appear until after the first true leaves emerge. In the second Chapter, 1, 2 and 3 mm thick TCL explants obtained from in vitro germinated plants were established on MS culture medium with Picloram (450 μM). It was observed that the TCL technique can efficiently induce somatic embryogenesis in oil palm. Genotypes and thickness of the explants used exert a strong influence on the induction of somatic embryogenesis. Explants with a thickness of up to 2 mm are more responsive. Genotypes also exert a strong influence on the induction of somatic embryogenesis, because shown important differences in the percentage of callus with somatic embryos and number of somatic embryos formed per callus. Finally, the third Chapter proposed a method for the vegetative reproduction of zygotic embryos (ZE) from interspecific backcrosses of palm oil by somatic embryogenesis, as well as to analyze genetically and epigenetically the regenerated plants through ISSR and AFLP markers. To this end, 195, 281 and 198 ZE from the respective interspecific backcrosses SQ150, SR78 and SR84 were cultured in vitro under the experimental conditions developed in Chapter 1, with minor modifications. From the regenerated plants, 36 individuals of three different zygotic embryos (considered as matrices) were randomly evaluated by ISSR and AFLP (MSAP) markers for possible genetic and epigenetic variations. After 36 months of cultivation, plant regeneration rates ranged from 6.2% to 34.2%. When the regenerants were evaluated by ISSR, it was possible to infer that regenerated individuals of ZE can present between 97.5% and 100% of similarity when evaluated within the same matrix that originated them (intrapopulations). However, when individuals are compared with regenerated plants from other ZE used as matrices, they showed genetic variability of more than 20%, that is, genetic similarity of only 80% (interpopulations). In the analysis performed by the AFLP (MSAP), 357 loci were amplified. In these amplifications, it was verified that 69% of these regions were sensitive to methylation and of these, 58% were caused by the gain of fragments, that is, by the hypomethylation of the genomic DNA.