Ayman Almerei

Abstract

Agrobacterium is widely considered, when suitably modified, to be the most effective vector for gene transfer into plant cells. For a long time, many cereals crops (monocotyledonous plants) were recalcitrant species to genetic modification, mainly as a result of their recalcitrance to in-vitro regeneration and their resistance to Agrobacterium infection. However, recently Agrobacterium-mediated transformation has been used to transform monocot crops such as maize (Zea mays) but with severe restrictions on genotype suitability. This study was carried out to evaluate the transformation amenability of 2 Syrian maize varieties and 2 hybrids in comparison with the hybrid line Hi II by the Agrobacterium tumefaciens-mediated transformation technique using a callus induction based system from immature zygotic embryos IZEs. A. tumefaciens strains EHA101, harbouring the standard binary vector pTF102, and the EHA105 containing the pBINPLUS/ARS:PpCBF1 vector were used. The effects of genotypes and the size of IZEs explants on callus induction and development were investigated. Results showed that callus induction and subsequent callus growth were significantly affected by the initial explant size. Calli induction from IZEs explants sized 1.5-2.00mm was 76%. Callus weight however decreased to 8.2g, compared with 11.7g of callus derived from IZEs >2.00mm. Callus induction ranged between 73.6-78.9% for varieties and hybrids respectively. Calli derived from varieties weighed significantly more than those initiated from the hybrids. Results demonstrated that Syrian maize genotypes were efficiently transformed via the A. tumefaciens strains but there was variation in transformation frequency. A transformation frequency of 3.7-4.2% was achieved for hybrids and varieties respectively confirming that the transformation frequency was genotype-dependent. The transformation frequency averaged between 3.2-5.6% for the EHA105 and EHA101 respectively. Fertile transgenic plants were regenerated from mature somatic embryos with an average regeneration frequency of 59.2 and 17% respectively for varieties and hybrids. Transgenic seeds of R0 and R1 progenies were produced from 74% of the outcrosses attempted and more than 98% of transgenic plants were normal in morphology. Fertile transgenic maize plants carrying the transferred gene CBF were produced using the Agrobacterium EHA105/PpCBF1 and these plants were shown to be more salt tolerant. Transient expression of the GUS gene was confirmed in transgenic calli, shoots, leaves, roots and floral parts of transgenic R0 and R1 progenies using histochemical GUS assays. The presence of the introduced bar and CBF genes in the genomic DNA of the transformants was confirmed by the PCR amplification. Further, the stable expression of the CBF and bar transgenes in the maize genome of transgenic R1 progeny was confirmed by qRT-PCR. The transformation protocol developed using an A. tumefaciens standard binary vector system was an effective and reproducible method to transform Syrian maize with an anti-stress gene in which fertile salt-resistant transgenic plants were routinely produced. This approach has great potential for development of Syrian maize breeding programmes for abiotic stress resistance for application in many areas in Syrian maize production.

Keywords

Genetic transformation, Agrobacterium, Abiotic Stress, Anti stress genes, Syrian maize, Drought, Salinity, Immature zygotic embryos

Document Type

Thesis

Publication Date

2016

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