Tissue culture and biolistic-mediated transformation of impatiens balsamina

Abstract

Tissue culture is the maintenance of fragments of tissue in vitro. In the beginning of this project, a procedure has been achieved for efficient regeneration system using cotyledonary explants. The 7 days old seedling cotyledon which showed the highest average number of shoots per explants during shoot induction was used in this study. The regeneration system of Impatiens balsamina was achieved using 1 mg/L BAP and 0.1 mg/L IAA within 8 weeks in culture. Plant transformation system has been defined as the stable incorporation and expression of foreign gene into plants. In this study biolistic technique was used for the transformation. The physical and biological factors of bombardment were optimized by monitoring the transient gusA gene expression. Using the optimal bombardment conditions, the explants were pre-cultured in 0.2 M mannitol and 0.2 M sorbitol for 16h prior bombardment. The explants were bombarded using pRQ6 containing hph gene conferring resistance to hygromycin and co-transformation was carried out using pRQ6 with pAHG11. The latter contain bar gene conferring resistance to Basta. The bombardment was carried out once at 28 Hg vacuum pressure, a helium pressure of 1100 psi, 9 cm of target distance and the plasmid concentration of 1.0 µg. The post-bombardment incubation time of 24h resulted in high gusA transient gene expression. Biolistic- mediated transformation of Impatiens balsamina was achieved with 18.3% transformation frequency. A total of 14 resistant plants in 75 mg/L hygromycin were GUS positive. PCR signal confirm the integration of hph gene into the genomic DNA of Impatiens balsamina.