Agrobacterium Mediated Transformation of Selected Maize Inbred Lines with pPZP200 towards Enhancement of Lysine and Methionine Content

Abstract

Maize (Zea mays (L.) is one of the most important cereals used both for human and animal consumption in the world. Despite its importance, maize is not a suitable single source of nutrition because it does not provide the essential amino acids lysine and methionine in sufficient quantities to meet the nutritional needs of humans and other animals. Lysine is a necessary building block for protein in the body while methionine is the body’s primary source of sulphur. Strategies to improve the nutritional quality of maize for high lysine and methionine have involved both genetic engineering (GE) and non-genetic engineering approaches such as marker assisted selection. Breeding is however laborious, lengthy and carries along undesired alleles. The objective of this work was to manipulate maize inbred lines towards enhancement of lysine and methionine content in the endosperms through Agrobacterium mediated transformation. Maize kernels mainly store proteins as ?, ?, ? and ? zeins. The immature embryos of three tropical maize inbred lines (TL18, CML216 and CML144) and a temperate line (A188) were transformed using Agrobacterium tumefaciens strain EHA101 carrying an expression cassette designed to up-regulate the Z10 protein for methionine enhancement as well as down-regulate the ? zein storage protein by RNAi. The T-DNA also contained P-zp22/6 as the promoter and the phosphinothricin acetyltransfarase gene (bar) used for selection of transformed tissue. Putative transformants were tested for presence of the transgene by PCR designed to amplify the P-zp22/6 promoter sequence. Calli survival frequencies were calculated as a percentage number of surviving calli in relation to the total number of embryos infected. These ranged from 2.89 % for TL18 to 9.11 % for A188. This data did not detect any significant difference (p>0.05) among the genotypes on the percentage of calli which survived. Transformation efficiency was calculated as a percentage of the number of PCR positive plants divided by the total number of embryos infected. This ranged from 0% for TL18 to 1.83% for A188. The data suggest the possibility of manipulating storage proteins and regenerating normal transgenic maize with normal kernels. Further work should involve gene expression assays for accumulation of ?, ? and ? prolamins in the kernels and southern blot analysis to confirm stable integration and the copy numbers of P-zp22/6 gene in the PCR positive plants.

Keywords: RNAi, Z10, Lysine, Methionine, pPZP200