Cloning Abiotic Stress Associated Genes and Agrobacterium tumefaciens Mediated Transformation of Selected Tropical Maize
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Date
2014-02-18
Authors
Abdalla, Rasha Adam Omer
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Abstract
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.
Description
Department of Biochemistry and Biotechnology, 191p. 2013, S 595 .A2