Genetic Engineering and Gene Transformation
Saeed Soheilivand; Amir Mousavi; Mohammadreza Safarnejad
Abstract
Sour lime (Citrus aurantifolia L.) is one of the most important woody plants is widely known for its recalcitrance to genetic transformation. We aimed herein to evaluate effective factors influencing the transformation efficiency and the reduction of chimeric transgenic shoots in sour lime. Epicotyl ...
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Sour lime (Citrus aurantifolia L.) is one of the most important woody plants is widely known for its recalcitrance to genetic transformation. We aimed herein to evaluate effective factors influencing the transformation efficiency and the reduction of chimeric transgenic shoots in sour lime. Epicotyl and internode explants were genetically transformed with different Agrobacterium tumefaciens strains e.g., LBA4404, GV3850, and GV3101, harboring the vectors pBI121 and pCAMBIA3301 containing β-glucuronidase (GUS) as a reporter gene. The effect of the following factors was evaluated: Agrobacterium concentration (OD600=0.3, 0.5 and 1), during inoculation (5 seconds, 10 minutes and 30 minutes), co-culture (2 and 3 days), and the selection regime (phosphinothricin at 1, 3, 5 and 10 mg/l and kanamycin at 25, 50, 75 and 100 mg/l). In following, transformation efficiency and the chimeric transgenic shoots rate were respectively confirmed by PCR and GUS assays. The results showed that Agrobacterium strain LBA4404, at the OD600 of 0.5, with 5 seconds (for epicotyl) and 10 minutes (for internode) inoculation at two-day co-culture period, were identified the most suitable treatments for both explants. The transformation frequencies ranged from 0.93% for internode on DKW medium containing 1.0 mg/l of phosphinothricin to 14.29% for epicotyl on DKW medium containing 50 mg/l of kanamycin. Inclusion of the high-level of selective treatments, improved the transformation rate through decreasing frequency escape and chimeric transgenic shoots. These findings provide novel insights into the appropriate procedure to constitute non-chimeric lime transgenic shoots.
Plant Disease and Biotechnology
M.R Safarnejad; M Basirat; M.A Ebrahimi; H Safarpour; S Nazari; B Mahmoudi; S Ataie Kachoie
Volume 2, Issue 3 , January 2013, , Pages 1-12
Abstract
Rhizomania is one of the most important Sugar beet diseases throughout the world. The disease is caused by beet necrotic yellow vein virus (BNYVV). The Polymyxa betae (Keskin) is the only natural transmitting agent of the disease between the plants. The fungus is an obligate parasite and could not be ...
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Rhizomania is one of the most important Sugar beet diseases throughout the world. The disease is caused by beet necrotic yellow vein virus (BNYVV). The Polymyxa betae (Keskin) is the only natural transmitting agent of the disease between the plants. The fungus is an obligate parasite and could not be cultured in the media, then detection of fungus is done normally by microscopic observation. In order to facilitate detection process, present study is done to develop specific antibodies against this fungus by applying recombinant protein. The Glutathione-S-transferase (GST) as a specific immunogenic protein is a critical enzyme expressed in zoospores, sporangia and resting spores and could be a good candidate to develop a serological test for P. betae. For this aim, the DNA region encoding fungal GST gene was isolated and cloned into pET28a bacterial expression vector. Large scale expression of the recombinant protein was performed in Escherichia coli (Migula). Purification was carried out by applying immobilized metal affinity chromatography under native conditions. The purified recombinant GST protein was used for immunization of rabbit. Purification of immunoglobulin was performed by affinity chromatography using protein A column. The purified antibodies were applied for efficient detection of infected plant in serological assays.
