Regeneration and transformation of Egyptian maize inbred lines via immature embryo culture and a biolistic particle delivery system

Abstract

A regeneration system was developed for elite Egyptain maize inbred lines using immature embryos as explants. This system proved to be highly genotype-dependent. Line Gz 643 was identified as the best line, revealing the highest regeneration frequency (42.2%). Addition of l-proline and silver nitrate to culture media greatly enhanced the formation of embryogenic type II callus and the regenerability of some of the tested lines. Transformation of the scutellar tissue of immature embryos from inbred line Gz643 was performed with the particle delivery system using a single plasmid carrying both the GUS and Bar genes (pAB-6) or by co-transformation with two plasmids, pAct1-F (GUS) and pTW-a(Bar). Different transformation parameters were evaluated, i.e. ostomic treatment, acceleration pressure, and number of shots. Osmotic treatment (0.25 M sorbitol + 0.25 M mannitol) along with the use of either acceleration pressure 1300 psi and one shot per plate (for co-transformation with pAB-6) or 1100 psi and two shots per plate (for transformation with pAct1-F and pTW-a) gave the best results, as expressed by the number of blue spots in the β-glucuronidase (GUS) assay. Stable transformation was confirmed in Ro transformed plants by means of histochemical GUS assay and herbicide application. PCR and Southern blot analysis proved the integration of the full-length genes in some of the transgenics.