<i>Agrobacterium</i>-mediated transformation of protocorm-like bodies in <i>Dendrobium</i> Formidible ‘Ugusu’

Abstract

Agrobacterium-mediated genetic transformation system was established in Dendrobium Formidible 'Ugusu' by inoculating PLBs with A. tumefaciens strain EHA101 (pIG121Hm) harboring hygromycin phosphotranferase (hpt) and neomycin phosphotranferase II (nptII) genes as selectable marker gene and β-glucuronidase (gus) gene as a reporter gene. For obtaining the optimum conditions for the transformation, several factors such as the stage of PLBs after subculture, bacterial concentrations, kind of inoculation medium, inoculation time and inoculation condition (with or without rotary shaking), were examined. After inoculation, PLBs were cocultivated for 3 days and then transferred for selection onto 2.5 g l −1 gellan gum-solidified ND medium containing 10 g l −1 maltose, 20 mg l −1 hygromycin and 20 mg l −1 meropenem. Hygromycin-resistant plantlets were regenerated from secondary PLBs after 4 months of selection. Transformation of these plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The highest transformation efficiency of 18.5% was obtained when PLBs 3 weeks after subculture were inoculated with 1 : 10 diluted bacteria (OD 600 ≈0.1) with liquid medium containing only 10 g l −1 maltose and 100 µM acetosyringone with shaking for 30 min.

Agrobacterium-mediated transformation of Dendrobium orchid with the flavanone 3-hydroxylase gene

“…When the incubation period was increased from 30 to 60 min, the transformation efficiency decreased from 18% to 3%-4% (Men et al, 2003). In Dendrobium Formidible 'Ugusu' , the highest transformation efficiency (12.2%) was achieved when PLBs were incubated with the bacterial suspension (OD 600 = 0.1) for 30 min (Phlaetita et al, 2015). A longer incubation time for Agrobacterium-mediated gene transformation has also been reported in other orchids, such as Vanda, for which it was found that a 4-h incubation period gave the highest transformation efficiency.…”

“…Three months after cultivation, the transformed orchids generated leaves and roots ( Figures 7D-7F). Successful Agrobacterium-mediated gene transformation into Dendrobium orchids has been infrequently reported (Men et al, 2003;Atichart et al, 2007;Phlaetita et al, 2015). The efficiency of Agrobacterium-mediated transformation depends upon several factors, such as the type of explants, strain and concentration of bacterial cells, type of inoculation medium, incubation and cocultivation time, the Agrobacterium-eliminating agent, and the method used to screen and regenerate the transgenic plants.…”