Genetic manipulation of commercial orchid hybrids in Malaysia

Tee C.S., . and Ong A.J., . and Wee C.Y., . and Marziah Mahmood, . (2005) Genetic manipulation of commercial orchid hybrids in Malaysia. [Proceedings Paper]

Different plasmid constructs were introduced into two commercial orchid hybrids Dendrobium Sonia and Oncidium Taka using the biolistic method. Each plasmid harbours different types of reporter gene GUS or GFP different promoter sequences CaMV35S ubiquitin1 act1 Emu as well as different antibiotic selectable marker genes hptII nptII. For the transformation events different orchid target tissues protocorm-like-bodies callus meristem-tip of in vitro inflorescenceswere compared. The suitability of different forms of antibiotic or herbicide selections hygromycin kanamycin geniticin paromomycin Basta and processes of selection in liquid vs solid medium were also tested. For the Dendrobium hybrid it was found that ubiquitin1 and act1 were better than other promoters tested. The antibiotic selection revealed that the orchid tissues for both hybrids were very sensitive to hygromycin at low concentrations 20-25 mgl. The study also showed that antibiotic selection of the orchid tissue carried out in liquid medium maintained on a rotary shaker at 100 rpm had more rapid toxic response 7-10 days compared to that on solid medium 1 month. All orchid tissues tested showed that they were suitable for transformation using the biolistic method. Transient expression of GUS was carried out according to a standard assay Jefferson et al. 1987 while GFP expression was viewed under the Leica MZ FLIII stereomicroscope using fluorescence GFP2/3 filter sets. The optimal expression of the reporter genes either GUS or GFP was highly dependent on the age of the tissues. Antisense constructs harbouring flower colour genes chalcone synthase and dihydroxy-flavonol-reductase were successfully introduced into the orchid tissues. Currently glasshouse assessments of the flower colour change are ongoing.

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Abstract

Different plasmid constructs were introduced into two commercial orchid hybrids Dendrobium Sonia and Oncidium Taka using the biolistic method. Each plasmid harbours different types of reporter gene GUS or GFP different promoter sequences CaMV35S ubiquitin1 act1 Emu as well as different antibiotic selectable marker genes hptII nptII. For the transformation events different orchid target tissues protocorm-like-bodies callus meristem-tip of in vitro inflorescenceswere compared. The suitability of different forms of antibiotic or herbicide selections hygromycin kanamycin geniticin paromomycin Basta and processes of selection in liquid vs solid medium were also tested. For the Dendrobium hybrid it was found that ubiquitin1 and act1 were better than other promoters tested. The antibiotic selection revealed that the orchid tissues for both hybrids were very sensitive to hygromycin at low concentrations 20-25 mgl. The study also showed that antibiotic selection of the orchid tissue carried out in liquid medium maintained on a rotary shaker at 100 rpm had more rapid toxic response 7-10 days compared to that on solid medium 1 month. All orchid tissues tested showed that they were suitable for transformation using the biolistic method. Transient expression of GUS was carried out according to a standard assay Jefferson et al. 1987 while GFP expression was viewed under the Leica MZ FLIII stereomicroscope using fluorescence GFP2/3 filter sets. The optimal expression of the reporter genes either GUS or GFP was highly dependent on the age of the tissues. Antisense constructs harbouring flower colour genes chalcone synthase and dihydroxy-flavonol-reductase were successfully introduced into the orchid tissues. Currently glasshouse assessments of the flower colour change are ongoing.

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Item Type:	Proceedings Paper
Additional Information:	Summary En
AGROVOC Term:	Genetic manipulation
AGROVOC Term:	Orchidaceae
AGROVOC Term:	Hybrids
AGROVOC Term:	Biolistics
Geographical Term:	MALAYSIA
Depositing User:	Ms. Norfaezah Khomsan
Last Modified:	24 Apr 2025 05:28
URI:	http://webagris.upm.edu.my/id/eprint/16651

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