Development of an efficient transformation system for Catharanthus roseus cell cultures using particle bombardment

We have developed an efficient direct DNA transfer procedure for the facile engineering of Catharanthus roseus cell cultures. Particle bombardment of callus derived from leaf material permitted rapid selection and establishme nt of transgenic cell lines. Transgenic callus were recovered at a frequenc y of between 60-80% of total callus bombarded with a single plasmid. Bombar dment using two separate plasmids resulted in a 25-60% frequency of transge nic callus recovered, up to 90% containing both input plasmids, Between 10- 20 g FW of transgenic material was produced within 3 months of bombardment, providing sufficient material for molecular and biochemical analyses. We d eveloped two complementary systems allowing selection on either hygromycin or kanamycin to permit re-transformation using plasmids carrying additional genes of interest. Use of leaf tissue as explant for transformation avoids time-consuming and labor intensive procedures involving suspension culture s. We provide molecular data on integration and expression of selected and non selected transgenes in a number of transgenic callus lines. Transgene i ntegration events for co-transformed plasmids were relatively simple, occur ring at one or two sites in the genome for most of the lines we analysed. M olecular analysis of callus resulting from co-transformation experiments us ing two different plasmids revealed that in nine of 10 putative transgenic lines we selected for analysis both plasmids had integrated into the genome . RNA gel-blot analysis and histochemical staining showed that an unselecte d transgene, gusA, was expressed in seven of the ten lines we analysed. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.