Routine utilization of green fluorescent protein as a visual selectable marker for cereal transformation

AbstractThe green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable marker-free transgenic coffee plants.

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Trypanosoma cruzi Coexpressing Ornithine Decarboxylase and Green Fluorescence Proteins as a Tool to Study the Role of Polyamines in Chagas Disease Pathology

Enzyme Research ◽

10.4061/2011/657460 ◽

2011 ◽

Vol 2011 ◽

pp. 1-10 ◽

Cited By ~ 11

Author(s):

Jeremías José Barclay ◽

Luciano Gastón Morosi ◽

María Cristina Vanrell ◽

Edith Corina Trejo ◽

Patricia Silvia Romano ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Trypanosoma Cruzi ◽

Chagas Disease ◽

Ornithine Decarboxylase ◽

Fluorescent Protein ◽

Selectable Marker ◽

Green Fluorescence ◽

Parasite Survival ◽

Green Fluorescent

Polyamines are essential for Trypanosoma cruzi, the causative agent of Chagas disease. As T. cruzi behaves as a natural auxotrophic organism, it relies on host polyamines biosynthesis. In this paper we obtained a double-transfected T. cruzi parasite that expresses the green fluorescent protein (GFP) and a heterologous ornithine decarboxylase (ODC), used itself as a novel selectable marker. These autotrophic and fluorescent parasites were characterized; the ODC presented an apparent Km for ornithine of 0.51 ± 0.16 mM and an estimated Vmax value of 476.2 nmoles/h/mg of protein. These expressing ODC parasites showed higher metacyclogenesis capacity than the auxotrophic counterpart, supporting the idea that polyamines are engaged in this process. This double-transfected T. cruzi parasite results in a powerful tool—easy to follow by its fluorescence—to study the role of polyamines in Chagas disease pathology and in related processes such as parasite survival, invasion, proliferation, metacyclogenesis, and tissue spreading.

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