Abstract

Constructs carrying visual reporter genes coupled with efficient promoters could facilitate the process of identification and selection of stable transformants in recalcitrant crops. Here, a novel construct utilizing a ribulose-1,5-bisphosphate carboxylase (RbcS) promoter combined with the green fluorescent protein (GFP)reporter gene to initiate very high expression of GFP in florist's chrysanthemum(Chrysanthemum morifolium Ramat.) was described. Based on this expression cassette, a new regeneration protocol using leaf discs as explants was developed for the Agrobacterium-mediated transformation of Chrysanthemum genotype ‘1581’, and a transformation efficiency of 7% was obtained. The expression of two different GFP constructs targeted to either cytosol or plastids was compared in transgenic lines. Both GFP constructs were expressed at such a high level that the green fluorescence dominated red fluorescence in the leaf tissues, allowing easy observation and microdissection of transformed tissues even without a GFP filter.Under normal light, plants with GFP targeted to plastids had a light green phenotype deriving from the high GFP expression. Quantitative reverse transcriptional PCR analysis showed that the plastid targeted construct with intron had significantly higher steady state transcript levels of GFP mRNA. This novel expression cassettemay allow direct visual selection of transformed tissues independent of antibiotic selection in a wide range of plant species.

Key words: Florist's chrysanthemum (Chrysanthemum morifolium), genetic transformation, GFP gene, RbcS promoter, RbcS targeting signal.

Abbreviation

BA, 6-Benzyladenine; GFP, green fluorescent protein; MS,Musrashige and Skoog’s medium; NAA, naphthalene acetic acid; NPTII, neomycin phosphotransferase; RbcS, ribulose-1,5-bisphosphate carboxylase.