Cloning Abiotic Stress Associated Genes and Agrobacterium tumefaciens Mediated Transformation of Selected Tropical Maize
Abdalla, Rasha Adam Omer
MetadataShow full item record
Drought and salinity are the main abiotic constraints of maize production. The genes confer tolerance to abiotic stresses can be isolated, cloned and introduced into important crops such as maize. The Annexin1 (AnnAt1) and Annexinp35 have been highly associated with water stress tolerance. AnnAt1 is induced by various stresses including water and oxidative stress and has been found to have peroxidase activity. Annexin p35 is a maize gene that also has peroxidase activity and plays a role in exocytosis, calcium transport and regulation. The Na+/H+ antiporter (NHX1) gene is involved in compartmentalization of Na+ into the vacuoles and therefore playing an important role in salt tolerance. However these genes have not been reported in transformation of tropical maize and its response to water stress. Annexinp35 has never been cloned and used in transformation of any crop. The objectives of this study was to isolate and clone the maize Annexinp35 gene and transform tropical maize with the Annexinp35, AnnAt1 and NHX1 genes conferring drought and salt tolerance via Agrobacterium tumefaciens and screening for stable integration and expression of the transgenes in T0 transgenic plants. An initial assessment of maize response to callus induction was performed using mature embryos, shoot tips and leaf segments and different levels of 2,4-Dichlorophenoxyacetic acid (2,4-D). AnnexinP35 gene was isolated from egg cells of maize and cloned into pNOV2819 vector, the AnnAt1 gene in pROK2 vector was sub-cloned into pNOV2819 vectors with it is promoter and terminator, the PMI gene from pNOV2819 vector was sub-cloned in to pCAMBIA-NHX1 vector which carries the salt tolerant gene NHX1 gene. Drought and salt tolerant genes were engineered into Sudanese maize genotypes, These genotypes are both striger resistant and most preferred by farmers in Sudan. The drought and salt tolerance genes were introduced to Sudanese maize using Agrobacterium tumefaciens method. The experiment was carried out using random complete block design. Transformation frequency and efficiency were assessed by using mannose as selectable agent. Transformation frequency and efficiency were found to be genotypic dependant. Transformation frequency and efficiency were evaluated for all the genotypes used in the study. Shoot tips gave the highest callus induction frequency among all the genotypes used while mature embryos gave the lowest callus induction frequency. The highest transformation frequency for the gene construct pCAMBIA-NHX1/PMI was observed in STR136 (8.03%). IL15 and Mojatamma-45 had TF of 2.05% and 1.75% respectively while the highest TE was observed in IL15 (1.13%). The highest transformation frequency for the vector pNOV2819- ASARZMANNp35 was observed in Hudiba-2 (31.78%) while the lowest TF was observed in Mojtamma-45 (5.38%). The highest TE was observed for Giza-2 (1.00%). The gene construct pNOV2819-AnnAt1 gave highest transformation frequency in the Inbred local-5 (23.98%) and lowest TF in IL1 (3.75%). The highest TE was observed Giza-2 (2.48%) for the same gene construct. Drought tolerant lines generated will be available to the maize breeders to transfer the trait to lines that have high yield but lack this trait.