In-Vitro Regeneration, Genetic Transformation and Evaluation of Maize (Zea Mays L.) For Resistance to Maize Lethal Necrosis Disease

Abstract

Maize (Zea Mays L.) is one of the most important cereal crops in the world and over 300 million people in sub-Saharan Africa depend on it for food. The majority of Kenyan population, approximately 85%, directly depends on maize for food. However, since the emergence of maize lethal necrosis disease (MLN), Kenya has experienced losses of about US$ 140 million annually. Most of the existing MLN management approaches are ineffective. Gene silencing through transgene induced ribonucleic acid (RNA) is simple and rapid, where small RNA molecules pair with messenger RNA (mRNA) eliciting nuclease degradation or silencing. The method has been adopted for plant disease management. The objectives of this study were to: optimize a regeneration protocol for the International Maize and Wheat Improvement Center maize lines (CML) CML442 and CML444 inbred maize lines; transform maize with the P1, HC-Pro, or VPg transgenes of sugarcane mosaic virus (SCMV), identify transgenics and confirm transgene integration via conventional polymerase chain reaction (PCR) and restriction digestion, respectively and assay for MLN response. Plant regeneration from immature embryos was tested at 0, 2, 3 and 4 mg/L of 2, 4-D in Murashige and Skoog (MS) media in a completely randomized design. The best in-vitro regeneration performing maize inbred line was transformed with the P1, HC-Pro, or VPg transgenes. The presence of transgenes was confirmed by PCR and restriction digestion with restriction endonucleases; AscI, BfmI and AsuII for the VPg, P1 and HC-Pro, respectively. Resistance for MLN was conducted via detached leaf assay (DLA). Disease severity was scored on a CIMMYT scale of 1-5. Both CML444 and CML442 were not significantly different at (p≤0.001) during regeneration with CML442 recording a mean of 8.90 as compared to 6.85 in CML444. The lines were significantly different at (p≤0.05) during rooting with CML444 recording a mean of 35. 67 as compared to 28.67 in CML442 leading to selection of CML444 for transformation downstream. Digestion products of 303, 227; 535 bp, 169 bp; 1222, 163 bp for VPg, P1 and HC-Pro genes were detected, respectively. The DLA assay indicated that the VPg transgenics had a minimum disease severity score of 1.44, followed by 3.82 and 4.01 for the P1 transgenic and HC-Pro transformants, respectively. Based on our findings, the VPg transgenic indicated that gene silencing through transgenic induced RNA could be the solution for MLN management