Presence and expression of the Ds-Red gene in the transgenic protocorm-like body of the Dendrobium cv. Burana White

Abstract

Four transgenic lines of the Dendrobium cv. Burana White containing the target DsRed gene were obtained following the microprojectile bombardment. Protocorm-like bodies (PLBs, etiolated), cut into 3-5 mm in size, were used as target material for bombardment. Gold particles (0.9 µm) coated by the mixed plasmids (pITB-DsRED with DsRed gene along with the plasmid pITB-BAR with bar gene) (Molar ratio 3:1, respectively) were bombarded into PLBs by the BIO-RAD PDS-1000/He. After 2-day co-bombardment, PLBs were cultured on the Knudson C (1946) selection medium (1 mg/l of BA) with 1-3 mg/l of phosphinothricin (PPT) for several selection cycles. The transient expression of the DsRed gene (2 days after bombardment) was confirmed with a positive result. The PCR analysis of the DsRed, bar genes showed the expected DNA bands - 350 bp, 500 bp, respectively. For the detection of the DsRED fluorescence, the transgenic PLBs (etiolated and green) were illuminated with a hand-held LED spotlight (for yellow-green light - 560 nm wavelength) and photographed by the CANON digital camera in combination with a red filter. Furthermore, the expression of the DsRED protein in PLB was also readily identifiable under day light/white fluorescent lamp. These results indicate that DsRed is a suitable reporter for genetic transformation of Dendrobium and this system could be used in fundamental research, especially the change of flower color due to the biochemical product of the transgene. This is the first result on genetic transformation of Dendrobium with the DsRed gene for generating transgenic PLBs which can be excited for emitting strong red fluorescence