Background

In vitro organogenesis is one of the key techniques associated with genetic transformation, as it determines the successful regeneration of transgenic plants after co-cultivation with bacteria. Therefore, the development of efficient regeneration protocols is the most critical step in developing genetic transformation. Protocols have been developed for several species of eucalyptus. In most of the studies cotyledon, hypocotyl and leaf segments of plants cultivated in vitro are used as explants [1]. Several eucalyptus functional genomics projects have used Populus species as a model for it counts with well established regeneration and transformation protocols. However, the use of Eucalyptus clones as model plants could be more adequate, if clones with high regeneration rates as those obtained for Populus could be found. The aim of this study was to evaluate several variables in the in vitro organogenesis from leaves of an Eucalyptus sp. clone maintained in vitro at Embrapa Forestry.

Methods

The experiments were performed at the Laboratory of Tissue Culture of Embrapa Forestry, Colombo, PR. Leaf explants were collected from in vitro grown plants maintained on MS medium [2] containing 30 g L-1 sucrose, 0.88 µM BAP, 0.05 µM NAA, 0.5 g L-1 and 7 PVP g L-1 agar. The youngest leaves were cut longitudinally and placed with the adaxial side facing the media. In the first experiment, the effect of 0.5 µM thidiazuron (TDZ) was compared with different concentrations of zeatin (2.28, 4.56, 9.12 and 13.68 µM). The plant growth regulators were added to the basic medium, composed of MS salts with half of nitrogen concentration (N/2), supplemented with vitamins of Morel and Wetmore, 30 g L-1 sucrose, 0.1 g L-1 myoinositol, 0.1 µMNAA and 7 g L-1 agar. The second experiment compared the effect of MS salts N/2, WPM [3] and B5 [4] on the same basic medium described above, containing 0.5 µM TDZ. In the third experiment the effect of different concentrations of TDZ (0.25, 0.5, 0.75, 1 and 2 µM) added to the basic medium, replacing the MS N/2 by WPM was evaluated. The explants were kept in growth chamber with controlled temperature at 23±2 C in the dark for four weeks, and then transferred to the same medium after 2 weeks. After this period the explants were transferred to basal medium with WPM, 20 g L-1 sucrose, 0.1 g L-1 myo-inositol, 5 µM BAP, 0.05 µM NAA and 7 g L-1 agar and placed under a photoperiod of 16 hours. The pH of all media was adjusted to 5.8 before autoclaving. After two months explants oxidation, callus formation, shoot formation in explants with callus and number of shoots per explants with callus were evaluated. Each treatment consisted of five Petri dishes with 10 explants. Data were analyzed by an analysis of variance. Comparisons between treatments were made by orthogonal contrasts.