Physical and Biologically Effective Parameters in Developing Transplastomic Tobacco Plants by Particle Bombardment Method using PDS-1000/He

Optimization of the effective parameters in the biolistic method for high-performance DNA transferring into the chloroplasts without tissue damage is a necessary requirement for developing transplastomic plants. Due to the relative similarity of the physical barriers at the entry of microcarriers into the nucleus and chloroplasts, the nuclear gus reporter gene is usually used, but the physical and biological parameters involved in plastid engineering have not been directly reported. In this study, to optimize gene transfer to tobacco chloroplasts, some parameters such as microcarrier type, DNA concentration, helium accelerating pressure, leaf arrangement and shooting distance, and pretreatment duration of explants before bombardment were investigated. After transferring the chloroplast's vector to explants, regeneration of transplastomic plants was performed in a selection medium containing 500 mg/l spectinomycin. The highest number of transplastomic plants (2.5 plants per bombardment) was obtained with Tungsten particles with a diameter of 0.7 μm, with 1 μg DNA/shooting, accelerating helium pressure 1100 psi, shooting distance of 6 cm and 24h pre-treatment period. Homoplasmic plants were obtained after three re-regeneration periods at the presence of both strepto/spectinomycin antibiotics. Molecular confirmation of the correct insertion of expression cassette at the targeted place of the genome and the confirmation of homoplasmy were performed using PCR analysis and southern blotting. Maternal inheritance of the transferred genes into the chloroplasts was confirmed by green and albino seedlings resulted from seed germination, product of reciprocal crosses between control and transplastomic tobacco plants.