Browsing by Author "Abdalla, Rasha Adam Omer"

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    Agrobacterium tumefaciens mediated transformation of Sudan maize genotypes using NPKI gene for enhancing drought stress tolerance
    (2011-11-29) Abdalla, Rasha Adam Omer; Jesse Machuka; Abdelbagi M. Ali
    Drought is one of the most important abiotic factor affecting maize production worldwide. Agrobacterium-mediated gene transfer technique has been established as a versatile way of improving important crops for tolerance to biotic and abiotic factors. Through this technique, the drought tolerance gene, NPKI, has been used in the transformation of temperate maize after its isolation and characterization from tobacco. Recovered transgenic events were observed to have enhanced tolerance to water stress. The accelerated adoption of the transformation technique in Africa, and indeed in Sudan, will depend on the ease with which transgenes of agronomic importance can be integrated into appropriate germplasms. This study aimed at screening important Sudanese maize inbred lines and open pollinated varieties (OPVs) for transformability via the integration of the NPKI gene. Eight inbred lines and three OPVs were evaluated. A188 was used as the standard inbred line check while KAT was used as the local OPV check. Freshly isolated immature embryos of maize were inoculated with Agrobacterium strain EHA101 harbouring the plasmid pSHX004 in LS infection media for 5 minutes and then co-cultivated on LS cocultivation media for 3 days. Embryos were then transferred to selection media supplemented with 250mg/l cefotaxime and 1.5mg/L bialaphos. After two weeks on this media, calli were subcultured on selection media containing 3.0 mg/L bialaphos for 4 weeks. Bialaphos resistant callus events were then transferred to maturation media supplemented 3mg/L bialaphos for 2 weeks before transferring to shooting media. Shoots were then transferred to rooting media. Plantlets with well-formed root system were transferred from the in vitro environment to green house for hardening. Hardened plantlets were transplanted to soil in the greenhouse and maintained till they set seeds. To confirm the presence of the transgene, PCR analysis was done on putative transgenic plants using the Bar primers. Out of a total of 4401 immature embryos from the 13 genotypes infected, 327 survived selection in bialaphos. Bialaphos resistant calli emerged 3-4 weeks after selection. IL3, IL15, Hudiba-2, IL1, IL38, Hudiba-1, A188 and KAT produced compact calli from their scutella surfaces while IL28, IL42, IL43, Mojtamaa-45 and IL16 established watery nonembryogenic calli. Statistically significant differences (p<0.05) were observed between the genotypes with respect to transformation frequency (TF). IL3 was identified as the most amenable to transformation with a TF of 31.7% and proved to be superior to A188, which recorded a TF of 5.82%. Hudiba-2 was identified as the most transformable OPV with a TF of 8.7% compared to that of 7.3% for KAT. ILI and Mojatamaa-45 proved to be poor responders to transformation with TFs of 2.5% and 1.7%, respectively. Putative transgenics were recovered from IL3, IL 15, Hudiba-2, ILI, IL38, Mojatamaa-45, A188 and KAT. The frequency of regeneration of bialaphos resistant shoots varied from 6.9% for IL38 to 100% for Mojtamaa-45. PCR analysis indicated a 540bp fragment in the DNA extracts from transgenic R, plants. Transformation efficiency (TE) was found to depend on the genotype used. The highest TE was observed for IL3 (3.7%), while the lowest TE of 0.0% was observed in IL42 IL43, IL16 and 11,28. Various abnormalities were observed in putative transformants including dwarfism, tussel seed and lack of ear. However, plants grew to maturity and were able to establish seeds in spite of these abnormalities. In conclusion, the inbred line IL3 and the OPV Hudiba-2 proved to be the most amenable Sudanese genotypes to A. tumefaciens-mediated transformation. Future research in maize improvement through biotechnologies such as tissue culture and genetic transformation should be focussed on these good responders.
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    Cloning Abiotic Stress Associated Genes and Agrobacterium tumefaciens Mediated Transformation of Selected Tropical Maize
    (2014-02-18) Abdalla, Rasha Adam Omer
    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.