Transient β-gus and gfp gene expression and viability analysis of microprojectile bombarded microspores of Brassica napus L.

Microprojectile bombardment of Brassica napus microspores was investigated based on transient gene expression of the marker genes beta-glucuronidase (gus) and green fluorescent protein (gfp). Biological and physical bombardment parameters to maximize gene delivery were established. Microspore viability after bombardment was also determined. Evaluation of 4 different promoters, sunflower polyubiquitin, napin A, 1' and CaMV-35S led to the use of the polyubiquitin promoter for standardization of bombardment parameters. This promoter showed the highest transient gene expression in early microspore stages. Developmental stage and quality of the microspores were the main factors found to influence transgene expression. The highest transient expression and viability frequencies were observed after bombardment of microspores incubated for 48 h or 72 h at 32 degrees C after isolation. Filtration of the 72 h-incubated microspores through cell strainers (sieves) retained only expanded and embryogenic microspores for bombardment. As a result of the enrichment of viable microspores, transient gene expression frequencies were considerably improved compared to microspores bombarded over filter papers or agarose plates. Under the best experimental conditions, transient GUS expression was observed in 0.17% of the total bombarded microspores. However, the drastic drop in microspore viability observed after bombardment was the largest drawback, resulting in a low regeneration rate of 0.06% microspore-derived embryos. The use of the marker gene, S65T-gfp, for microspore transformation is promising, since it allows detection and potential selection of viable transgenic microspores and their regeneration into embryos.