Evaluation of parameters which affect the agrobacterium mediated genetic transformation of eucalyptus grandis. / Avaliação de parâmetros que influenciam a transformação genética do Eucalyptus grandis via Agrobacterium.

AUTOR(ES)

Alexander de Andrade

DATA DE PUBLICAÇÃO

2001

RESUMO

The research project aimed the establishment of a method for genetic transformation of Eucalyptus grandis; an important and widely planted forestry tree in Brazil. Two techniques of transformation were tested: agrobiolistic of explants with accumulation of meristematic cells and SAAT ( sonication-assisted Agrobacterium mediated transformation ). Both systems of transformation aim to produce micro wounds in the explants in order to increase the Agrobacterium penetration in the tissues. In the case of agrolistica the explant was previously submitted to micro projectile bombardment, followed by bacteria inoculation. On the other hand in the SAAT technique the explante is submitted to short periods (few seconds) of sonication together with the bacteria. Transformation was evaluated by measuring the expression of the reporter gene uidA which codes the ß-glucuronidase enzyme. Several parameters were tested for agrobiolistic such as, gas pressure (helium), flight distance of micro projectiles, gene expression of the uidA, co-cultivation temperature. The highest values of ß-glucuronidase were observed for 1350 PSI, 9.5 cm from target and 26O C for co-cultivation. A histological analyze was carried out to check it the meristematic tissues were being transformed. The results showed that the meristematic tissues were localized in the surface of the explante. The meristem is formed by the dedifferentiation of epidermal cells and the first layers of the cortical parenchyma indicating its exogenous origin. The histological location of the reporter gene uidA showed that the expression occurs only in cells already differentiated, and not in meristematic cells. For SAAT experiments a clone was previously selected for favorable characteristic of transformation: high rate of regeneration, susceptibility of selective agents and to Agrobacterium transformation. The highest rate of ß-glucuronidase activity were observed for 60 s of sonication; higher sonication time reduced the efficiency of regeneration. Additional sonication, following a preliminary sonication of explante , in the presence of the bacteria, improved the efficiency of transformation. The results provided evidence the SAAT is a viable technique for Eucalyptus transformation via Agrobacterium.

ASSUNTO(S)

explante vegetal melhoramento genético vegetal bacteria biolística bactéria fitopatôgenica genética florestal genetic transformation transformação genética explants parâmetro genético eucalyptus eucalipto

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