In this study, we report an optimization of particle bombardment transformation system for Centella asiatica callus. A total of eight parameters affecting the genetic transformation system were optimized using the synthetic green fluorescent protein (sGFP) as a reporter driven by the CaMV 35S promoter. The results indicated that DNA delivery conditions of 9-cm target distance, 1,100 psi helium pressure, 1.0 µm gold particles size, 27 mmHg chamber vacuum pressure, 2 times number of bombardment, spermidine as precipitation agent, 60 h postbombardment incubation time and 2 µg plasmid DNA concentration were optimal for C. asiatica callus transformation. The expression of sGFP was monitored using fluorescence microscope and further confirmed using RT-PCR. This optimized genetic transformation system is applicable for rapid transient gene analysis and transgenic C. asiatica production.
We explored the use of the medicinally important plant Centella asiatica for expression of hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) strain AF2240. HN protein is the principal target for subunit vaccine development against NDV. The full-length HN gene was cloned into a plant expression construct driven by the CaMV 35S promoter and C-terminal fusion of green fluorescence protein (GFP) as reporter system. The recombinant expression construct was transformed via particle bombardment into C. asiatica callus. Transformants were screened using GFP and selected on MS medium supplemented with 15 mg/l hygromycin. The ~1.8 kb HN mRNA transcript was detected on the putative transformants using RT-PCR. The presence of HN protein expression was further confirmed through dot blot analysis using anti-NDV chicken serum. Here we report, for the first time, the use of a novel medicinal plant as a new platform for HN protein expression.
JavaScript jest wyłączony w Twojej przeglądarce internetowej. Włącz go, a następnie odśwież stronę, aby móc w pełni z niej korzystać.