Temperature, acetosyringone, virulence genes and wounding effects on Agrobacterium-mediated transformation efficiency

Abstract

Several conditions were evaluated to determine their impact on Agrobacterium-mediated transformation efficiency. Temperature and acetosyringone (AS) effects were first studied in tobacco, as a plant model system and then applied to cotton transformation. In tobacco, AS significantly increased transformation rates that resulted in stable transformed plants. Four temperatures were evaluated during the cc-cultivation period to determine the most appropriate temperature for T-DNA transfer and integration into the plant genome. Even though 19°C was reported in the literature as the optimal temperature for Agrobacterium-mediated gene transfer, 25°C was statistically significantly different as compared to 15°C, 19°C or 32°C and produced the highest number of transgenic tobacco plants. The superior treatments established in tobacco transformation were applied to Agrobacterium-mediated transformation of the cotton shoot apex. Explant survival rates in selection medium were recorded and used to compare two temperatures (19°C and 25°C) and the effects of AS to induce the vir genes. After approximately 6-7 wk in selection medium, the percentage of surviving exhalants established that 25°C was the best temperature for cotton transformation, either with or without AS. Two wounding methods and the addition of a super-virulent plasmid were tested in cotton shoot apex transformation experiments. Their effects were evaluated using the green fluorescent protein (GFP) as an in vivo reporter gene to determine the early transformation patterns. Both removal of a leaf primordial and sanitation were examined. Transient GFP expression did not reveal any major benefit of the treatments on the transformation pattern. In contrast, when additional copies of virG and virE genes were inserted into Agrobacterium, a considerable increase in the number of fluorescence spots per exploit was evident. This was the first time GFP gene was used in cotton. Its expression was reliable, and the system was successfully established. Since GFP has several advantages as a reporter gene over the traditional GUS gene and herbicide resistance genes, it would allow the development of a more accurate selection process and therefore it would speed up the optimization of the shoot apex method.