Agrobacterium-Mediated Transformation of Elite Kenyan Maize Germplasm with CRY3A Gene for the Control of the Larger Grain Borer and Maize Weevil
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Date
2013-10-18
Authors
Taracha, Catherine Ongecha
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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