Optimisation of biological parameters for transformation of Oncidium Taka via the biolistic method
2005
Wee, C.Y. | Marziah Mahmood | Kadzimin Saleh | Tan, C.S.
The biolistic method was used in the genetic transformation of Oncidium Taka using protocorm-like bodies and calli as target tissues. In this study, biological parameters were optimized by observed expression of green fluorescent protein as transient expression. Bombardment was carries out using 3 mg gold microcarriers coated with 5ug of DNA into both target tissues that were pre-cultures on the same day to prevent a decrease in the quality and quantity of transient expression. Strong green fluorescent signals were easily detected using a Leica Stereo-fluorescence System MZ FLIII. The parameters optimized were as follows: expression of five different types of modified green fluorescent protein gene, maximum expression time after bombardment, pre-culture period of target tissue prior to bombardment, and age of target tissues. The effects of the green fluorescent protein expression on different target tissues were also examined. In this study, synthetic GFP-TYP mutant, P35S-sgfp-TYG-nos, gave the best transient expression in both target tissues. The highest transient target green fluorescent protein expression was observed two days post-bombardment. In comparison to the single and multi-cellular system, protocorm-like-bodies had significantly higher green fluorescent protein expression compared to calli. The physiological stages of the PLBs and calli may also influence the degree of expression. From this study, seven- and fourteen-day cultures of both calli and protocorm-like-bodies, respectively, had the highest transient expression.
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