Agrobacterium-Mediated Transformation of the Oryza sativa Thaumatin-Like Protein to Canola (R Line Hyola308) for Enhancing Resistance to Sclerotinia sclerotiorum

Document Type: Research Paper

Authors

1 Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

2 Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

Abstract

Background: Canola is an agro-economically oilseed crop. Yield loss due to fungal disease of stem rot caused by Sclerotinia sclerotiorum is a serious problem in canola cultivation. Thaumatin-like proteins are large groups of the pathogenesis-related proteins which provide resistance to the fungal infection in response to invading pathogens and play a key role in plant defense system.
Objectives: Transformation of the rice tlp into canola via Agrobacterium-mediated transformation and evaluation of the antifungal activity of the expressed TLP in the transgenic events on the S. sclerotiorum growth was subject to investigation.
Materials and methods: The canola (R line Hyola308) was used for transformation experiment. The vector, pBITLPRA1, was used for the stable transformation. The PCR and southern blotting techniques were used to confi rm transgene’s presence in the transgenic canola events. Antifungal activity of transgenic plants was evaluated by the radial diff usion and spore germination assays. T2 transgenic plants were evaluated by the intact leaf inoculation method in greenhouse assay.
Results: In this study, pBITLPRA1 construct containing tlp gene was introduced into canola and the transformed plants were verifi ed by PCR. The glucanase activity of tlp gene in T0 generation was measured and transgenic plants with high activity were assessed by Southern blot analysis to confi rm the copy number of the gene. Also, antifungal activity of the single copy T0 transgenic plants against Sclerotinia sclerotiorum was evaluated by radial diff usion and spore germination assays. In greenhouse assay, evaluation of T2 transgenic plants by the intact leaf inoculation method demonstrated that following the infection with S. sclerotiorum, there was a signifi cant reduction in the lesion’s diameter in transgenic lines compared to the non-transgenic ones.
Conclusions: These results revealed that expression of TLP has an inhibitory eff ect against fungus compared to nontransgenic plants both in vitro and in vivo (i.e., greenhouse condition). These transgenic lines could be used as the additional sources of disease resistance for canola breeding program.

Keywords

Main Subjects