A highly efficient and reproducible protocol was developed to obtain transgenic Alstroemeria plants by combining Agrobacterium tumefaciens with friable embryogenic callus (FEC). To develop this transformation method, factors such as infection time, cocultivation period, effect of acetosyringone (AS), different dilution concentrations of the bacterium and temperature during cocultivation were evaluated. A protocol was developed in which transient GUS expression activity was observed ranging from 25% to 55% out of the cocultivated FEC cultures, when FEC cultures were infected for 30 min with 50 lM AS, 1:10 dilution of bacteria, and then cocultivated at 24°C in the dark for 7 days with Agrobacterium strain LBA4404 (pTOK233) that carried gus, nptII and hpt genes. Seven independent experiments produced a total of 1300 transformed somatic embryos with shoots from 3.5 g of FEC. Of these germinated embryos, 50% developed into plants in vitro. Thus, on average, 500 mg of FEC infected with A. tumefaciens produced approximately 80-100 transgenic plants within 6-8 months via a selection process with 2.5-20 mg L 21 hygromycin. Additionally, transformation was also performed with Agrobacterium strain AGL1 (containing the uidA and ppt genes), and this showed that luciferase-based selection was less detrimental to the transgenic lines than was herbicide-based selection. The transformation efficiency was 18.6% for the luciferase-based selection and 7.6% for the PPT-based selection, although with luciferase-based selection, more false positives were obtained (about a quarter of the lines were escapes). The nptII and uidA genes were detected by polymerase chain reaction analysis in nine of the 19 tested lines. The results indicate that the system developed here can be used as an alternative to particle bombardment of Alstroemeria.

This is the first successful report of Agrobacterium-mediated transformation in Alstroemeria by infection of FEC (Friable Embryogenic Callus) lines and leaves with axil tissues. Of the transformation methods, particle bombardment and Agrobacteriummediated transformation have been widely used to transfer foreign DNA into the plant genome. Especially, Agrobacterium tumefaciens efficiently infects most plants. Most monocotyledonous plants, including Alstroemeria, are recalcitrant to A. tumefaciens infection. Therefore, it is essential to develop an efficient, reliable protocol for Agrobacterium-mediated transformation in Alstroemeria. In order to achieve this aim, different hygromycin concentrations in selection medium were tested and compared in FEC explants and leaves with axil tissues using Agrobacterium strain LBA4404 (pTOK233). As a result, 20 mg/l hygromycin was a proper concentration to select for both FEC lines and leaves with axil tissues. Using this concentration, transformed lines were obtained. Moreover, FEC lines showed at least 2.5 times higher rate in the survival rate, and 5 times higher rate in transient GUS gene expression than those of leaves with axil tissues. The protocol, seems to be a promising method for the transformation of the monocot Alstroemeria with genes of interest in the near future.