Maize Transformation for Development of Stem Borer
(Sesamia cretica) Reistance
Using Bt Genes

Maize is one of the major cereal crops in Egypt with a total cultivated area of approximately 2 million feddans (Feddan = 4200 sq.m). Maize fields are commonly infested with three species of stem borers, of which Sesamia cretica is the most damaging. It causes losses in yield varying from one season to another. Sources of resistance in the maize germplasm have not been identified. New biotechnology techniques to generate genetically improved plants resistant to insect attack are available. They are based on isolating, characterizing and cloning a gene from Bacillus thuringiensis (Bt) responsible for the production of an insect controlling protein. Therefore, the introduction of genetically modified maize plants with insect resistance (Bt toxin gene) will be of great value not only in maximizing yield, but also in reducing the use of chemical pesticides.

OBJECTIVES

•  Develop an efficient regeneration system for Egyptian maize genotypes from immature embryo callus and cell suspension cultures.

•  Continue sequencing Bt genomic clones isolated at AGERI from Egyptian strains.

•  Develop bioassay procedures for screening Bt toxin effects against maize stem borers.

•  Conduct kill curve studies for maize cell line(s) using different concentrations of antibiotic and/or glyphosate herbicide to identify the minimum level needed to be used for selection of putatively transgenic lines in vitro.

•  Initiate transformation of maize callus and/or cell suspensions using reporter (GUS) and selectable marker genes with the biolistic bombardment system.

•  Construct a plasmid containing a wild type Bt coding sequence and a series of chimeric genes containing codon-modified Bt coding sequences to continue transforming in vitro regenerable maize genotypes.

•  Confirm the integration of genes (at the genomic) level with Southern blot hybridization.

•  Confirm gene expression at the mRNA level by Northern blot hybridization.

•  Confirm gene expression at the protein level by protein western analysis using polyclonal antibodies for Bt.

•  Preliminary evaluation of transgenic plants against maize stem borers in the laboratory and greenhouse.