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

• Published in: Plant science (Limerick) Vol. 140; no. 2; pp. 179 - 188
• Main Authors: Hilliou, Frédérique, Christou, Paul, Leech, Mark J.
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 01.01.1999; Elsevier Science
• Subjects: Agronomy. Soil science and plant productions; Biological and medical sciences; Biotechnology; CATHARANTHUS ROSEUS; CELL CULTURE; Co-transformation; CULTIVO DE CELULAS; CULTURE DE CELLULE; Fundamental and applied biological sciences. Psychology; GENE TRANSFER; Genetic engineering; Genetic engineering applications; Genetic technics; GENETIC TRANSFORMATION; Genetics and breeding of economic plants; Methods. Procedures. Technologies; Particle bombardment; Plant breeding: fundamental aspects and methodology; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; TRANSFERENCIA DE GENES; TRANSFERT DE GENE; TRANSFORMACION GENETICA; TRANSFORMATION GENETIQUE; Transgenic animals and transgenic plants; Transgenic callus; TRANSGENIC PLANTS; Co-transformation; FW, fresh weight; GUS, β-glucuronidase; Catharanthus roseus; MS, Murashige and Skoog medium; Transgenic callus; Particle bombardment; Cell culture; Microprojectile bombardment; Genetic transformation; Explant; Plant leaf; Medicinal plant; Apocynaceae; Alkaloid; Dicotyledones; Angiospermae; Genetic improvement; Genetic engineering; Spermatophyta; Secondary metabolism
• ISSN: 0168-9452; 1873-2259
• DOI: 10.1016/S0168-9452(98)00225-8

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 establishment of transgenic cell lines. Transgenic callus were recovered at a frequency of between 60–80% of total callus bombarded with a single plasmid. Bombardment using two separate plasmids resulted in a 25–60% frequency of transgenic 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 developed 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 cultures. We provide molecular data on integration and expression of selected and non selected transgenes in a number of transgenic callus lines. Transgene integration events for co-transformed plasmids were relatively simple, occurring at one or two sites in the genome for most of the lines we analysed. Molecular analysis of callus resulting from co-transformation experiments using 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 unselected transgene, gusA, was expressed in seven of the ten lines we analysed.