Production of fertile transgenic wheat plants by laser micropuncture

Abstract

A modified, non-damaging, protocol for the production of fertile transgenic wheat (Triticum aestivum L. cultivar Giza 164) plants by laser micropuncture was developed. The new homemade setup secures the transformation of as many as 60 immature embryo-derived calli (10000 cells each) in less than one hour using a UV excimer laser with two dimensional translation stages, a suitable computer program and a proper optical system. Five-day-old calli were irradiated by a focused laser microbeam to puncture momentarily made self-healing holes ( approximately 0.5 microm) in the cell wall and membrane to allow uptake of the exogenous DNA. The plant expression vector pAB6 containing bar gene as a selectable marker for the herbicide bialaphos resistance and GUS (uidA) gene as a reporter gene was used for transformation. No selection pressure was conducted during the four-week callus induction period. Induced calli were transferred to a modified MS medium with 1 mg l(-1) bialaphos for regeneration, followed by selection on 2 mg l(-1) bialaphos for rooting. Three regenerated putative transgenic events were evaluated for the integration and stable expression of both genes and results indicated that this modified procedure of laser-mediated transformation can be successfully used in transforming wheat.

Optoperforation of single, intact Arabidopsis cells for uptake of extracellular dye-conjugated dextran

“…Cultured cells of Brassica napus have been shown to remain viable following UV (343 nm, 15 ns) laser irradiation followed by uptake of bizbenzimide-labelled pBR322 plasmid, with cytoplasmic streaming visible one hour post treatment and similar survival rates [3]. Cultured wheat cells were also transformed with a gusA expression vector by a UV laser (6 ns, 308 nm with pulse energies up to 13 mJ) with confirmation of transformation seen in 3 out of 600 samples [4].…”

Virus-like particles production in green plants

“…Direct DNA transfer techniques range from transfection of protoplast by electroporation or chemical methods to laser micropuncture of isolated cells [26,27]. The most common method is particle bombardment, also known as the biolistic method, a combination of biological and ballistic.…”

Single-cell optoporation and transfection using femtosecond laser and optical tweezers

“…These parameters could change between the different cell lines. Different parameters related to the cell's viability, such as optimum laser exposure power, exposure time, puncture hole diameter, and healing time should be optimized before transfection [20][21][22][23][24][25][26][27][28]. This punctured hole is clearly visible through the CCD.…”

“…Successful transfection results were achieved by laser-assisted optoporation [20][21][22][23][24][25][26][27][28][29][30][31][32][33] of the cell membrane, which intakes external DNA present in the medium through diffusion. In the case of laser-assisted optoporation of the cell membrane, nanosecond or femtosecond laser pulses have also been used to create pores or to alter the permeability of the cell membrane at a specific time to make it possible to intake external substances such as plasmids.…”