Plastid transformation of tomato (Solanum lycopersicum L.) with HIV-1 p24 gene by biolistic particle delivery system
Keywords:Chloroplast transformation, Solanum lycopersicum L., HIV-1 p24 gene, aadA gene, promoter RRN.
Plastid transformation in tomato (Solanum Lycopersicum L.) is carried out using vector carrying HIV-1 p24 gene and a selection of aadA gene, spectinomycin for resistance to antibiotics, and streptomycin. We used the hepta adaptor to replace the rupture disk retaining cap and microcarrier launch assembly. Three days after bombardment, the leaves and cotyledons were cut into small pieces each of 3 × 3 mm and placed on medium MS salts and B5 vitamins containing 2.0 mg/l Zeatin, 0.2 mg/l IAA, 50 mg/l spectinomycin for leaves, and medium MS salts and B5 vitamins containing 2.0 mg/l BAP, 1.0 mg/l IAA, 0.5mg/l Kinetin, 50 mg/l spectinomycin for cotyledons. Selected further time on the medium containing spectinomycin was up to 300 mg/l. PCR analysis of aadA and HIV-1 p24 genes of the tomato plastid transformation lines showed the presence of DNA amplification with the corresponding 250bp and 700bp. Southern blot analysis confirmed that the foreign genes were inserted into the tomato plastid genome. The result indicated the presence of HIV-1 p24 in the tomato plastid genome with the presence of DNA band of 5.8 kb. However, it is heteroplasmic transformant because it has a DNA band of 3.5 kb of wide type plant.