Genetic transformation of two species of orchid by biolistic bombardment

Abstract

We report here the transformation of two species of orchid, Dendrobium phalaenopsis and D. nobile, by biolistic bombardment. Calli or protocorm-like bodies (PLBs) were used as target explants. Gold particles (1.0 µm) coated with plasmid DNA (pCAMBIA1301) encoding an intron-containing β-glucuronidase gene (gus-int) and a hygromycin phosphotransferase (hpt) gene were introduced into the PLBs or calli using the Bio-Rad PDS-1000/He Biolistic Particle Delivery System. Calli and PLBs were then chopped up and precultured in 1/2-strength MS medium supplemented with 0.4 M mannitol for a 1-h osmoticum treatment before bombardment. Immediately after bombardment, the calli and PLBs were transferred to 1/2-strength MS medium without mannitol for recovery. Putatively transformed plantlets were obtained by selection and regeneration on medium supplemented with 30 mg/l hygromycin. The highest efficiency of transformation was obtained when selection was conducted at 2 days post-bombardment. For D. phalaenopsis and D. nobile, respectively, about 12% and 2% of the bombarded calli or PLBs produced independent transgenic plants. Integration and expression of the transgenes were confirmed by Southern hybridization and Northern hybridization. No nontransformed plants were regenerated, indicating a tight selection scheme. However, separate incorporation of the gus gene and the hpt gene was observed, and in one transgenic line the gus gene was integrated into the genome of the transgenic plant, but not expressed.

Perspectives on MADS-box expression during orchid flower evolution and development

“…Although stable transformation mediated by Agrobacterium (Belarmino and Mii, 2000; Yu et al, 2001) and biolistic bombardment (Chia et al, 1994; Men et al, 2003) have been adapted to Phalaenopsis , Oncidium and Dendrobium , among others, their relatively low efficiency and long regeneration time to obtain flowering plants has limited their application in this area.…”

Evaluation on the effectiveness of 2-deoxyglucose-6-phosphate phosphatase (DOGR1) gene as a selectable marker for oil palm (Elaeis guineensis Jacq.) embryogenic calli transformation mediated by Agrobacterium tumefaciens

“…Delaying exposure to a selection agent is often carried out in order to provide time for the single transgenic cell to divide a few times and increase its ability to express the transgene in the presence of selection pressure as demonstrated in peanut and maritime pine (Ozias-Akinns et al, 1993; Alvarez and Ordás, 2013). In orchid, when selection was delayed to 30 days no transformants were recovered, and the highest transformation efficiency was obtained when selection was initiated 2 days after transformation (Men et al, 2003b). The transformed EC were exposed to medium containing 400 mg l -1 2-DOG as a selection agent.…”

A fresh method of DNA transformation to the seeds irradiated by 60Co without the use of antibiotic selection

“…Now, considering the main transformation methods to plants, there are other problems besides the use of some antibiotic genes as markers. For agrobacterium-mediated (Kemal et al, 2003) and biolistic bombardment methods (Men et al, 2003;Bohorova et al, 1999), first, only the plants which erected a regeneration system could be transformed. Secondly, there were very significant differences on the transformation ratio between different plant species and between different explants.…”