Marker-free lines of phytase-transgenic Brassica napus show enhanced ability to utilize phytate

Abstract

Through regeneration of 30 selected Brassica napus cultivars a non-genotype-specific and highly efficient regeneration system was established using two-step tissue culture. Plant regeneration frequencies from explants of cotyledons with petioles were 75–100% in different cultivars. Co-transformation was used to transfer exogenous genes with two expression vectors. LBA4404 Agrobacterium strains harboring the pBINphy (containing the phytase gene) and pBINBar (containing the selectable bialaphos resistance marker gene bar) were mixed at an equal ratio (1:1) and used to coinfect B. napus. A total of 10 transgenic B. napus lines expressing the bar and phytase genes were obtained, with a phytase/bar cointegration frequency of 50% in T0 plants. B. napus doubled haploid (DH) lines were obtained by microspore culture. Among these lines, a total of 14 phytase-transgenic DH B. napus lines free of selectable markers were obtained based on PCR screening. Western blotting showed that the phytase protein was normally expressed in the transgenic B. napus seeds. Both hydroponic and medium experiments showed that phytase-transgenic B. napus, but not nontransgenic control, could grow normally by utilizing phytate as the sole phosphorus (P) source. The phytase activity in the seeds of transgenic B. napus was approximately 3–20-fold higher than that in negative control seeds. Furthermore, the inorganic P content in the seeds of transgenic B. napus was also 2–6-fold higher than that in nontransgenic B. napus recipient seeds. Thus, the phytase-transgenic B. napus lines were developed that can utilize organic P and exhibit improved P uptake and utilization. The selectable marker free lines of phytase transgenic B. napus which can express and secrete phytase, grow normally by utilizing phytate as the sole phosphorus (P) source.