Transgene Integration Complexity and Expression Stability Following Biolistic Or Agrobacterium-Mediated Transformation of Sugarcane.

Sugarcane (Saccharum spp. hybrids) is one of the most productive crops and is extensively utilized for table sugar or biofuel production. Both agrobacterium-mediated transformation or biolistic gene delivery have been successfully used in the past for efficient gene transfer to sugarcane. Recent optimizations of biolistic gene transfer include reducing the amount of DNA and/or removing the vector backbone prior gene transfer. These modifications resulted in reduced complexity of transgenic loci and improved performance of transgenic plants. A direct comparison for biolistic transfer of minimal expression cassettes and agrobacterium-mediated gene transfer to sugarcane will be presented here.  Callus derived from 6 weeks culture of immature leaf whorl cross sections of commercial cultivar CP88-1762 was used as target for either biolistic gene transfer of the minimal nptII expression cassette or Agrobacterium- mediated gene transfer of the nptII expression cassette in five independent experiments.   Selection of transgenic events followed the same concentrations and types of selective agents for both transformation procedures. Real-time PCR for nptII (TaqMan®) and NPTII immuno-chromatography confirmed 279 transgenic lines from Agrobacterium-mediated transformation and 234 lines from biolistic gene transfer. Copy numbers of the nptII transgene were determined by TaqMan® real-time PCR and Southern blot. 20 lines, including 10 single copy lines and 10 multiple copy lines, from each gene transfer method were selected to propagate vegetative progenies. NPTII ELISA was carried out for these 40 lines for both primary transgenic plants (V₀) and their vegetative progeny (V₁). We will discuss the transformation efficiency, frequency of single copy integration, level and stability of expression following both gene transfer systems.