Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla

Abstract

This study aimed was to establish genetic transformation parameters for the hybrid Eucalyptus grandis X Eucalyptus urophylla, mediated by Agrobacterium tumefaciens as well as perform the cryopreservation of shoot tips for the same hybrid, aiming storage and conservation. The organogenic hybrid calluses were subjected to different transformation process, vacuum infiltration (4 to 8 minutes at 400 mmHg), sonication (15 and 30 seconds at a frequency of 40 kHz), combined and isolation. At the same time, were evaluated three concentrations of acetosyringone (100 μM, 200 μM and 300 μM) and three temperatures of cocultivation (19° C, 22° C and 25° C). Among the studied parameters, the mean value of the transient expression was obtained when using the infection method by vacuum infiltration for 4 minutes with mean transient expression of GUS activity (4,4), the addition of 100μM of acetosyringone mean transient expression (0.76), and co-cultivation at a temperature of 19° C with higher average transient expression (2.6). For the cryopreservation process was pre-determined regeneration medium of shoot tips using different concentrations of BAP (0,14 μM; 1,42 μM) and BAP (0,14 μM) in combination with IAA (5,10 μM), after 30 days it was evaluated the percentage of plant regeneration, calluses formation and vitrified plants. To cryopreservation was performed pre-cultivation of shoot tips on MS medium supplemented with 0.2 M sucrose for 24 hours and 0.5 M sucrose for 24 hours and were evaluated six times of exposure to PVS2 (20, 40, 60, 80, 100 and 120 minutes) and subsequent immersion in liquid nitrogen, after 30 days it was evaluated percent survival and after 60 days it was evaluated the percentage of shoots. Histological and ultrastructural observations of the apices were made in different stages of the cryopreservation process, and after one week of recovery, through light and scanning microscopy, of which measurements were made using plasmolysis image-Pro Plus software. The concentration of BAP 0,14 μM best promoted regeneration rates (80%) without the presence of vitrified plants. Cryopreservation was successful using 0.2 M and 0.5 M sucrose in the precultivation and exposure to a cryoprotectant for 60 minutes, providing no irreversible damage cells, resulting in higher survival rates (66%). The histopathological and ultrastructural analysis of the shoot tips were made during the cryopreservation process for the treatment of exposure for 60 minutes was found to increased cell integrity without the presence of picnotics nucleus with low plasmolysis rates when compared with treatments exposed to longer times (80 and 120 minutes) to the PVS2.