Optimization Of Various Bombardment Parameters In Biolistic Mediated Wueat(Triticumaestivum L. Var. Kenong199) Genetic Transformation

Biolistic mediated method is one of the two mainly employed ways in wheat genetic transformation.although Biolistic mediated wheat genetic transformation made remarkable progress In recent decades, butthe transformation efficiency is low because of its large genomic repeats and poor regeneration. In addition,wheat varieties (genotype) used for biolistic transformation is still very limited. Of the many parametersinfluencing the final transformation efficiency, gold particle size, target distance and acceleration pressureplay important roles. The objective of this study is to optimize the bombardment parameters and establishan efficient and stable biolistic transformation system of wheat (Triticum aestivum L.) variety Kenong199.In total,12different combinations of parameters including gold particle size, target distance andacceleration pressure were tested and optimized, and obtained the following results:1. Different parameters have a major impact on GUS transient expression. the average GUS transientexpression per embryo was43.6±2.1when the gold size is0.6μm, significantly higher than1.0μm of13.9±1.4. when the target distance increased to8.5cm, the average GUS transient expression per embryos was11.2±1.4, significantly lower than5.5cm of55.9±4.8. the average GUS transient expression per embryosunder high acceleration pressure (1100psi) was significantly reduced relative to650psi and900psi. The0.6μm gold,5.5cm target distance,650psi acceleration pressure combination had good performance withthe GUS transient expression per embryos of84.2±7.3.2. Different combination of parameters had different effects on the regeneration of calli.Embryogenesis efficiency of12parameter combinations were generally greater than84.9±8.8%, up to98.0±1.5%. But the regeneration efficiency were significant different among treatments. The0.6μm gold,5.5cm target distance,650psi acceleration pressure combination had good performance with theregeneration frequency of68.7±2.1%, the highest for all treatments, whereas the lowest of only23.3±3.8%.3. In total,12different combinations of parameters including gold particle size, target distance andacceleration pressure were tested and optimized, and resulted in a production of28independent transgeniclines,of which14transgenic plants were obtained in one treatment of0.6μm gold,5.5cm target distance,650psi acceleration pressure combination. The efficiency of final transformation through the aboveoptimized parameters combination was averaged at2.66%, with the highest reached at5.8%in oneparticular treatment. 4. Stable integration of GUS gene and its expression were confirmed by Southern blot analyses andGUS assay. Southern blot analysis demonstrated that the GUS gene in some transgenic lines were notexpressed due to the high copy numbers of this gene integrated into the genome which might lead to genesilencing.