PHD-Department of Biochemistry and Biotechnology

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Browsing PHD-Department of Biochemistry and Biotechnology by Author "Gethi, J."

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    Enhancement of Drought Tolerance in Tropical Maize Through Silencing of Poly (ADP-Ribose) Polymerase-1 Gene
    (2014-08-19) Matheka, Jonathan Mutie; Machuka, J.; Runo, Steven; Gethi, J.; Anami, S.
    In most of the production areas under maize, mild drought can significantly reduce yields while severe drought can sometimes completely destroy an entire plantation. Consequently, the livelihood of farmers is affected due to reduction in their incomes leading to poverty. Currently, very few highly transformable maize genotypes such as B73, W22, A188 and CM216 are used in genomic studies and genetic engineering of traits such as drought tolerance. Following trait enhancement through genetic engineering, consumer uptake of the transgenic product may be hindered due to presence of undesirable genetic elements such as selectable marker genes (SMG). Therefore generation of transgenic plants free of SMG is important to make them biosafe. The objective of this study was to generate SMG-free maize plants having an artificial microRNA (amiRNA) targeting the poly (ADP-ribose) polymerase 1 (PARP1) gene and assess transgenic plants for tolerance to severe oxidative and drought stress. In earlier studies using arabidopsis and rapeseed, the PARP1 gene silencing approach was demonstrated to enhance drought tolerance through preservation of cellular energy and reduced damage by reactive oxygen species. In this study, maize PARP1 gene silencing constructs (amiRNA-PARP1) were cloned in the same T-DNA region as the pmi or the bar SMG or placed in a separate T-DNA region for cotransformation of plants. The cotransformation vectors were first validated in tobacco before use in transformation of different maize genotypes. Maize transformation was achieved by co-cultivation of immature embryos with Agrobacterium tumefaciens harboring the amiRNA-PARP1 construct. Transgenic plants were assessed for downregulation of the PARP1 gene using qRT-PCR. The effect of PARP1 gene downregulation on drought tolerance was also assessed. Out of 13 genotypes evaluated, two (TL03B6754A-20 and TL03B6757-68) were found to be highly regenerable and were chosen for recovery of transgenic plants using either the PMI/mannose or bar/PPT selection system. Using the PMI/mannose selection system, the two maize genotypes were found to be highly transformable, averaging transformation frequencies (TF) of 48% and 34.16%, respectively. The TF for the control genotype CML216 was 32.19%. The TF under PPT selection` for the inbred lines CML216, TL03B 6757-68 and TL03B 6754A-20 was 26.16%, 14.81%, and 21.69% for pMarkfree3.1 and 27.22%, 32.10% and 36.32% for pMarkfree3.2, respectively. A qRT- PCR analysis conducted on six amiRNA-PARP1 transgenic lines revealed that the expression of the PARP1 gene was reduced in plants exposed to methyl viologen-induced oxidative stress. However, the level of PARP1 gene expression was higher in non- transformed plants. Plants with reduced expression of the PARP1 gene were tolerant to oxidative and drought stress indicated by higher chlorophyll content, relative water content, growth and biomass as well as reduced anthocyanin accumulation, compared to non-transformed plants. In conclusion TL03B6754A-20 and TL03B6757-68 genotypes are highly responsive to transformation. Therefore these inbred lines extend the pool of highly transformable genotypes available for genomic and trait improvement studies. In addition the cotransformation binary vectors developed here (pMarkfree3.0, pMarkfree2.1 or pMarkfree2.2) are applicable in any plant to produce SMG-free plants containing one or more transgenes of interest. In the long term, the drought tolerant transgenic maize plants produced in this study can help stabilize maize yields and increase production during water stressed conditions. This will impact targeted farmers and their households by increasing their incomes thereby improving their livelihoods.