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Kenyatta University (2011)

Agrobacterium tumefaciens mediated transformation of Sudan maize genotypes using NPKI gene for enhancing drought stress tolerance

Abdalla, Rasha Adam Omar

Titre : Agrobacterium tumefaciens mediated transformation of Sudan maize genotypes using NPKI gene for enhancing drought stress tolerance

Auteur : Abdalla, Rasha Adam Omar

Université de soutenance  : Kenyatta University

Grade : Master 2011

Résumé
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.

Mots clés : Corn—Breeding//Plant biotechnology//Plant gene isolation

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