Propagação in vitro de Eucalyptus microcorys e metodologias para a investigação do rejuvenescimento e revigoramento de tecidos

Abstract

Most woody species experience morphological, physiological and biochemical changes during the transition from the juvenile to the adult phase, especially with regard to the potential for cloning, growth vigor and adventitious rooting of propagules. Due to the lack of studies that address the propagation of Eucalyptus microcorys, and to the difficulty in rescuing and cloning adult donor plants using non-destructive techniques, this thesis aimed to promote in vitro propagation of E. microcorys, and to investigate the rejuvenation and/or reinvigoration of tissues. In the first chapter, a protocol via micropropagation using a donor plant aged over 40 years was developed. In the second chapter, a micropropagation protocol through indirect organogenesis was developed to evaluate the interaction of plant growth regulators in juvenile tissues. In the third chapter, possible indications of rejuvenation and/or reinvigoration of tissues propagated by different techniques, were investigated. In overall terms, the protocol via micropropagation, using epicormic buds, were suitable for in vitro cultivation, presenting an establishment rate of 40.55%, and multiplication of 820 explants until the 11th subculture. In the elongating phase, the best results were observed in WPM media with 0.05 mg.L-1 of BAP

+ 1 mg.L-1

of NAA and 0.05 mg.L-1

of BAP + 1 mg.L-1

of NAA + 1 mg.L-1

of AIB. In the rooting and acclimatization phase two models of microvessels (M2 with volume = 2.6 cm3; and M3 with volume = 5.3 cm3) presented the best results, which formed normal adventitious roots and of direct connection with the vascular cambium. In the stages of the indirect organogenesis, the callogenesis averaged 81.38% of callus formation in explants. In the bud induction phase, the explants from the cotyledon presented the highest mean values, while in the elongating phase, the explants from the hypocotyl tissue presented the best results the evaluated characteristics. Regarding the rooting and acclimatization phase, three models of microvessels (M4 with volume = 2.6 cm3; M6 with volume = 5.3 cm3; and M7 with volume = 4.9 cm3) presented the best results, which formed normal adventitious roots directly connected to the vascular cambium. Through molecular tests, confirmed the successful cloning of plants resulting from the micropropagation protocol by the ISSR technique, and a low genetic diversity of the microplants of the indirect organogenesis protocol. The micropropagation protocol through indirect organogenesis was efficient for the production of E. microcorys microplants, which requires as an explant the hypocotyl tissue grown in culture medium supplemented with 1 mg.L -1 TDZ, 1 mg.L

-1 TDZ + 4 mg.L

-1 NAA or 3 mg.L

-1 TDZ + 2 mg.L

-1 NAA. In the investigation of possible indications of rejuvenation and/or reinvigoration of tissues, materials resulting from plant production techniques were used: the seminal, grafting, micropropagation with epicormic shoots and indirect organogenesis. It was concluded that the individuals propagated via grafting presented different results with the other propagation techniques, but their behavior possibly does not match with greater rejuvenation and/or reinvigoration of the tissues. By the total protein test (TCA/Acetone/Phenol) two distinct regions were found in the samples obtained by grafting. The highest enzymatic activity of the peroxidase test was observed in the sample via micropropagation by epicormic shoots, demonstrating an indication of tissue rejuvenation. Regarding by pXRF, the best responses were detected for the distinction of materials propagated from the elements K, Ca, Zn, Fe, Mn, Si. However, new studies are recommended in order to seek other methods that have similarities with the responses to rooting in forest species and thus optimize the rescue in materials with different ontogenetic stages.