An efficient protocol for Agrobacterium-mediated transformation and regeneration of Campanula medium (Canterbury bells) based on leaf disc explants

Abstract

Campanula medium is an important ornamental plant known for its blue and bell-shaped flowers and a good model for studying flower colour formation and modification. The major outstanding obstacle preventing the developing of genetic modification and improvements in C. medium is a missing transformation method. For the first time, an effective and reliable in vitro regeneration and Agrobacterium-mediated transformation system has been established in this study. Different types and combinations of plant growth regulators (PGRs) were investigated for shoot regeneration by using leaf explants from three different cultivars. A high regeneration rate (50% for cv. ‘Blue double’, 58% for cv. ‘White double’ and 44% for cv. ‘Pink double’) was achieved on MS-based medium supplemented with 11.5 μM thidiazuron (TDZ) and 2.3 μM 1-naphthaleneacetic acid (NAA). In total, the Agrobacterium strains ABI, AGL1 and GV3101:pMP90, transformed with a binary plasmid harbouring NPTII and GFP gene cassettes, were evaluated for plant transformation, while various factors affecting the transformation efficiency were also identified. The highest transformation rate of about 22% was obtained from explants pre-cultured on regeneration medium and infected with AGL1. However, transgenic shoots were produced by all three Agrobacterium strains studied and confirmed by PCR and GFP fluorescence microscopy. Campanula medium regeneration and genetic transformation methods were established while various factors affecting the transformation efficiency were identified such as pre-culture treatment of leaf explants and different Agrobacterium strains.