( .Comparative Study of Transformation Efficiency , Between Gene Gun and Laser Microbeam Cell Surgery in Wheat (Triticum aestivum L

Abstract

Biotechnological methods especially, genetic engineering, tissue culture and protein engineering are a complement to breeding programs for the improvement of wheat productivity. The most successful approach to wheat transfonnation is the gene gun, i.e. bombardment of tissue derived from immature embryos followed by selection for resistance to bialaphos herbicide as conferred by the bar gene. It was speculated that the laser micropuncture has steadily developed as a method to perform selective subcellular microsurgery in human cells. A highly focused laser beam could be used as an "optical" microbeam to produce tiny submicrometer self-healing holes momentarily made in the cell wall and cell membrane to facilitate the uptake of exogenous DNA into cultured cells of wheat. In the present work, two transformation approaches were investigated for their efficiency in transfonning the cv. Giza 164 of wheat (Triticum aestivum), namely gene gun and laser microbeam cell surgery using two model genes, i.e. GUS and bar in comparison. Results showed that the two methods showed successful gene transfer. Histochemical GUS assay, PCR, restriction endonuclease analysis ofPCR products, Southern, ELISA as well as TEM confirmed gene presence and expression and evaluate the efficiency. Plants transformed by laser microbeam cell surgery showed transformation frequency over 4.5%, while gene gun technique showed efficiency of about 1.1%. Molecular analyses showed that laser microbeam cell surgery can be efficiently used in transforming wheat. Moreover, mechanical ultradamages using TEM could be recorded as a first study of its kind, showed that gene gun system was strongly affecting cell organelles, components, cell wall and cell membrane (40-70%) more than laser microsurgery (<I 0%). The later allowed self-healing of cell wall and membrane of transformed cells. The present work confirmed that the laser microbeam cell surgery could be considered as a more efficient, less damaging protocol over the gene gun method in transfonning wheat.