OPTIMIZATION OF FACTORS AFFECTING THE Agrobacterium tumefaciens- MEDIATED TRANSFORMATION OF Eucalyptus saligna

ABSTRACT This study aimed to evaluate the effect of factors that may affect the genetic transformation of cotiledonary explants of Eucalyptus saligna mediated by EHA105 strain of Agrobacterium tumefaciens. The vector pBI121 carrying gus gene under control of 35S CaMV promoter was used. The effect of the following factors was evaluated: explant pre-culture, use of different antibiotics and presence of acetosyringone (AS) in co-culture media. An antioxidant solution was also used during excision, containing ascorbic acid (250mg.L-1), citric acid (25mg.L-1) and PVP-40 (1g.L-1). Pre-culture of the explants before the co-culture with bacteria was done over a 4-day period in MS culture medium supplemented with 4.4µM BAP and 2.7ìM NAA. After theco-culture period, three concentrations of kanamycin (12.5;25 and 50mg.L-1) combined with 300mg.L-1 Augmentin® in the culture medium were tested The influence of the antibiotic was also evaluated by keeping the explants in a medium containing 50mg.L-1 Km and 300mg.L-1 Augmentin® or 500mg.L-1 cefotaxime. It was concluded that Augmentin® stimulates organogenesis, that a Km concentration of 12.5mg.L-1 allows selection of explants transformed with gus gene and, finally, the addition of AS (50ìM) to the liquid and solid co-culture media has a positive effect on gus gene expression. Moreover, the use of an antioxidant solution during cotyledon excision is dispensable and the pre-culture of the explants has no effect on bud regeneration or gus gene expression. A transformation efficiency of 1.5% was reached. RESUMO Esse trabalho teve como objetivo avaliar o efeito de vários fatores que podem afetar a transformação genética de explantes cotiledonares de Eucalyptus saligna mediante co-cultura com a cepa EHA105 de Agrobacterium tumefaciens. Foi utilizado o vector pBI121 carregando o gene repórter gus sob controle do promotor 35SCaMV. Foi estudado o efeito dos fatores a seguir: pré-cultura dos explantes, uso de diferentes antibióticos e presença de acetosiringona (AS) nos meios de co-cultura. Uma solução contendo ácido ascórbico (250mg.L-1), ácido cítrico (25mg.L-1) e PVP-40 (1g.L-1) foi utilizada no momento da excisão dos explantes. A pré-cultura dos explantes antes da co-cultura com a bactéria foi de 4 dias, em meio de cultura MS suplementado de 4,4µM BAP e 2,7ìM ANA. Após a co-cultura três concentrações de canamicina (12,5; 25 e 50mg.L-1) combinadas com 300mg.L-1 de Augmentin® no meio de cultura foram testadas. O efeito do Augment