Bioengineering Drought Tolerance in Tropical Maize (Zea Mays L.) through Inducible Expression of Isopentenyltransferase Gene

Abstract

Drought is one of the major abiotic constraints contributing to low productivity in maize. In tropical region, it causes grain yield losses of as high as 70% while complete crop failure is also common depending on the severity of drought. Drought diminishes crop productivity mainly by causing premature leaf senescence. It is now possible to delay drought induced leaf senescence in order to enhance tolerance to drought and stabilize crop yield through bioengineering. The ipt gene codes for isopentenyltransferase (IPT) enzyme, which catalyzes the rate- limiting step in the biosynthesis of cytokinin (CK) and enhances tolerance to drought by increasing the foliar level of CK that delays drought-induced leaf senescence in transgenic crops. This study was designed to genetically transform locally adapted elite and commercial tropical maize genotypes with ipt gene to develop drought stress tolerance through Agrobacterium tumefaciens-mediated genetic transformation. Ten maize genotypes adapted to Ethiopian and the Eastern and Central African (ECA) countries were evaluated for in vitro regeneration ability using immature zygotic embryos as explants. Six genotypes (Melkassa-2, Melkassa-6Q, [CML387/CML176]-B-B-2-3-2-B [QPM], CML395, CML442 and CML216) were identified as the best regenerating ones having potential for improvement through genetic transformation. Subsequently, the ipt gene was sub-cloned into the pNOV2819 binary vector to take advantage of the pmi gene as plant selectable marker and mannose as selective agent. The pNOV2819 binary vector carrying the ipt gene was introduced into the Agrobacterium strain EHA101 which was subsequently used to transform immature zygotic embryos obtained from the six genotypes. Among the six genotypes studied, transgenic plants were successfully regenerated in Melkassa-2 and CML216 with regeneration efficiency of 87.5 and 59.6%, respectively. Transgenic plants were analyzed using PCR, Southern blot and RT-PCR. Based on PCR results, transformation efficiencies were found to be 97.4 and 100% for Melkassa-2 and CML216, respectively, indicating stringency of the pmi/mannose based selection system for maize transformation. Southern blot analysis indicated stable integration of the transgene into the genome of CML216 with 2-3 copy numbers in five independent events. In drought assay carried out in the glasshouse transgenic plants expressing the ipt gene maintained higher leaf relative water content (RWC) and total chlorophyll concentration during the drought period and produced significantly higher grain yield, major yield components and root dry matter compared to the non-transgenic plants. The ipt gene was observed to improve drought tolerance in tropical maize by delaying drought induced leaf senescence. It was concluded that the transgenic line developed can be further tested for tolerance to drought under contained field trials. Furthermore it can be used in breeding programs to improve drought tolerance in other commercial tropical maize genotypes through conventional breeding.