A co-bombardment DNA delivery method to introduce foreign genes into Dendrobium Sonia 17

Tee C.S., . and Marziah Mahmood, . and Mariam Abdullah, . and Tan C.S., . (2005) A co-bombardment DNA delivery method to introduce foreign genes into Dendrobium Sonia 17. [Proceedings Paper]

PLBs-induced calli identified as types A and B of Dendrobium Sonia 17 were used as target tissues for genetic transformation work. Plasmid constructs 35S-SGFP-TYG-nos p35S and pSM CHS or pSM DFR were co-bombarded into both types of calli using the biolistic method PDS 1000 / Helium Biolistic Particle Delivery system. The p35S was used as it carries a sgfp gene driven by the 35S promoter. Visual observation of the green fluorescent protein GFP expression could be detected using a fluorescent microscope LEICA III. Such non-destructive assay could be used to access transient gene expression of transformation events leading to isolation of stable transformants. Both pSM CHS and pSM DFR carry a hygromcycin resistant hptII gene which allows selection of transgenic tissues resistant to hygromycin. The pSM CHS plasmid carries an antisense gene version of chalcone synthase CHS while pSM DFR carries an antisense gene version of dihydro-flavanol 4-reductase DFR. Both were used for blocking the endogenous CHS and DFR genes expression in the plant such that different flower colours would result from the transgenic plant. Three lines of putative transformants were obtained after more than 4 months of selection on medium containing hygromycin. They were from 3 independent co-bombardment events with p35S and pSM DFR plasmids using type A callus as target tissue. The results showed that GFP expression could be detected in all 3 lines of bombarded and selected tissues. However there were tissues that survived on selection medium without expressing GFP. Further molecular analyses are required to further test these transformants. A combination of GFP visual observation and antibiotic selection was effective for screening of transgenic tissues.

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Abstract

PLBs-induced calli identified as types A and B of Dendrobium Sonia 17 were used as target tissues for genetic transformation work. Plasmid constructs 35S-SGFP-TYG-nos p35S and pSM CHS or pSM DFR were co-bombarded into both types of calli using the biolistic method PDS 1000 / Helium Biolistic Particle Delivery system. The p35S was used as it carries a sgfp gene driven by the 35S promoter. Visual observation of the green fluorescent protein GFP expression could be detected using a fluorescent microscope LEICA III. Such non-destructive assay could be used to access transient gene expression of transformation events leading to isolation of stable transformants. Both pSM CHS and pSM DFR carry a hygromcycin resistant hptII gene which allows selection of transgenic tissues resistant to hygromycin. The pSM CHS plasmid carries an antisense gene version of chalcone synthase CHS while pSM DFR carries an antisense gene version of dihydro-flavanol 4-reductase DFR. Both were used for blocking the endogenous CHS and DFR genes expression in the plant such that different flower colours would result from the transgenic plant. Three lines of putative transformants were obtained after more than 4 months of selection on medium containing hygromycin. They were from 3 independent co-bombardment events with p35S and pSM DFR plasmids using type A callus as target tissue. The results showed that GFP expression could be detected in all 3 lines of bombarded and selected tissues. However there were tissues that survived on selection medium without expressing GFP. Further molecular analyses are required to further test these transformants. A combination of GFP visual observation and antibiotic selection was effective for screening of transgenic tissues.

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Item Type:	Proceedings Paper
Additional Information:	Summary En
AGROVOC Term:	DENDROBIUM
AGROVOC Term:	DNA
AGROVOC Term:	GENES
AGROVOC Term:	BIOLISTICS
AGROVOC Term:	GENETIC TRANSFORMATION
Geographical Term:	MALAYSIA
Depositing User:	Ms. Norfaezah Khomsan
Last Modified:	24 Apr 2025 05:28
URI:	http://webagris.upm.edu.my/id/eprint/16655

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