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Co-transfer of gfp,
CHS and hptII genes into Oncidium Sharry Baby
PLB using the biolistic gun
Ng Chea Yee1, Janna Ong Abdullah1*,
Maziah Mahmood2, Nazir Basiron3
1Department
of Microbiology, Faculty of Biotechnology and Biomolecular
Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang,
Selangor Darul Ehsan, Malaysia.
2Department
of Biochemistry, Faculty of Biotechnology and Biomolecular
Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang,
Selangor Darul Ehsan, Malaysia.
3Malaysian
Institute for Nuclear Technology Research (MINT), Bangi,
43000, Kajang, Selangor Darul Ehsan, Malaysia.
*Corresponding author: E-mail:
janna@biotech.upm.edu.my. Tel: 603-89466697. Fax:
603-89430913.
Abbreviations:
CaMV, Cauliflower Mosaic Virus; CHS, chalcone synthase;
hpt II, hygromycin
phosphotransferase type II; sgfp, synthetic green fluorescence protein;
PLB, protocorm-like-body; PCR, polymerase chain
reaction.
Accepted
25 June, 2008 |
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This study was aimed at assessing the biolistic method of
co-transforming non-linked genes on separate gene cassettes
into Oncidium Sharry Baby’s protocorm-like-body (PLB).
An expression vector containing a synthetic green
fluorescence protein (sgfp) gene driven by the
Cauliflower Mosaic Virus (CaMV) 35S
promoter and another vector containing the antisense
chalcone synthase (CHS) and hygromycin resistant (hpt
II) genes, were successfully co-bombarded into Oncidium
Sharry Baby PLB. Six critical parameters (PLB size, time
course of gfp transient expression in PLB, DNA
concentration, PLB age, presence of spermidine and CaCl2
in the DNA-microcarrier precipitation, and duration of PLB
on fresh medium prior bombardment) were optimized based on
transient gfp expression. One month after
bombardment, the PLB were subjected to 5
mg/ml
hygromycin selection for 8 months. A total of 137
regenerated putative transformants were randomly selected
and verified by polymerase chain reaction (PCR) analysis.
The results indicated the presence of the transgenes gfp,
hptII and antisense CHS in 28, 61, and 11% of the
selected putative transformants, respectively.
Key
words:
Biolistic gun, co-transformation, orchidaceae, protocorm-like-body. |
