Genetic Transformation of Farmer Preferred Tropical Maize Varieties and Inbred Lines using Drought Tolerance Conferring Genes Isolated from Xerophyta Viscosa
Seth, Miccah Songelael
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Maize supports lives of more than half of the population in Africa yet its production is extremely affected by drought. Yield losses of up to 70% are frequently attributed to drought stress. However, in extreme cases, total yield loss is often experienced. Low maize productivity may persist given the worsening situation of changing climate, coupled with erratic rainfall, prolonged drought spells and rising temperatures. Narrow genetic base of drought tolerant traits in available germplasm is another existing limitation in breeding for drought tolerance. Existing breeding strategies to develop maize capable of surviving drought can be complemented with recent biotechnology tools including genetic engineering. Plants such as Xerophyta viscosa Baker that have unique ability to survive extreme drought conditions have in the past provided useful genes and promoters that may find application in crop improvement against drought stress. For example, Xerophyta viscosa peroxiredoxin 2, (XvPrx2) and Xerophyta viscosa SAP1, (XVSAP1) genes have previously been isolated and characterised from Xerophyta viscosa. The XvPrx2 gene encodes a type II peroxiredoxin that scavenges for excess reactive oxygen species whereas the XVSAP1 gene encodes for an integral membrane protein that plays a role in stabilizing membrane integrity during dehydration stress. It was hypothesized that since ROS sequestration and maintenance of membrane intergrity are critical during drought stress, XvPrx2 and XVSAP1 genes could be useful in developing transgenic drought tolerant maize. The present study assessed the regeneration ability of selected maize germplasms adapted to Eastern and Central African region and the most regenerable ones were targeted for transformation. Plant expression vectors for genetic transformation contained phosphomannose isomerase gene that allowed the use of mannose as selective agent. One inbred line maize (CML144) and one open pollinated maize variety (Staha) were transformed using Agrobacterium-mediated transformation. Ten and 6 transgenic lines were recovered from CML144 and Staha maize, respectively using XvPrx2 gene and six transgenic lines were recovered from CML144 maize using XVSAP1 gene. To confirm the success in genetic transformation, polymerase chain reaction, Southern blotting and reverse transcription PCR were used to analyse transgenic maize. Transgenic maize plants were then subjected to drought stress assays that compared the performance of both transgenic and non-transgenic plants under dehydration. Transformation frequencies for CML144 and Staha transformed with XvPrx2 gene averaged at 12.9% and 23.9%, respectively. Transformation frequency for CML144 transformed with XVSAP1 gene was 45.2%. Stable transgene integration was revealed in transgenic T1 plants with low copy numbers ranging from 1 to 3. RT-PCR in T1 plants confirmed the expression of gene transcripts in transgenic maize plants under drought stress. Under drought stress, relative water content in transgenic CML144 and Staha plants were significantly higher than in non-transgenic maize. Analysis of chlorophyll contents under drought stress revealed relative stability of chlorophylls in transgenic CML144 and Staha compared to non transgenic maize plants. The transgenic plants designated CMl144-XvPrx2, Staha-XvPrx2 and CML144-XVSAP1generated in this study demonstrate great ability to tolerate drought stress under controlled laboratory conditions. However, further assessement of these transgenic plants under confined field conditions need to be conducted prior to availing the seeds to national agricultural research systems for integration into the breeding programs. The transgenic drought tolerant maize developed from this work can be grown in drought prone areas of Eastern and Central Africa.