Agrobacterium-Mediated Transformation of Elite Kenyan Maize Germplasm with CRY3A Gene for the Control of the Larger Grain Borer and Maize Weevil

Abstract

In Kenya maize (Zea mays L.) is the most important staple food. Owing to its predominant role, food security in the future cannot be achieved without major increases in cereal production. An efficient in vitro regeneration and transformation system holds a great potential for genetic improvement of maize against production constraints. This study was conducted with the objective of assessing the regenerative capacity, genetic transformation of Kenyan maize genotypes and efficacy of transformed maize in controlling maize pests. Six Kenyan inbred lines and four CIMMYT lines and their single crosses were evaluated for their in vitro response on three different media (MS, N6 and N6CL). The embryogenic callus induction and regeneration capacity was higher on MS than on N6 basal salts. Plant regeneration was influenced by genotype. Transformation experiments were carried out using Agrobacterium tumefaciens strain EHAI01 containing pTF 102 binary vector harbouring a GUS gene. The transformation frequency was highest in 104 (15.2%) and lowest in QPM. The highest transformation efficiency was recorded in inbred T04, 104 and their crosses . with CML 216 and ranged 2.4% to 3.0%. The transgene was detected in all the maize genotypes using GUS assays, and PCR. Maize genotypes were transformed using a Bacillus thuringiensis tenebrionis gene Cry3A. The highest transformation frequency was recorded in three inbred lines, H04, T04 and 104 (9.3%, 12.2% and 13.4% respectively). The transformation efficiency ranged between 0.5% to 2.5%. PCR and RT-PCR amplification of the Cry3A gene, and the l)AS-Elisa confirmed the presence of the gene To,T1 and T2 generations. 'Insect bioassays established that transgenic maize provided protection against the larger grain borer and maize weevil This study established a reproducible regeneration and transformation system for tropical maize, which can be used in a pest management programme