Kontturi, J., R. Osama, X. Deng, H. Bashandy, V. A. Albert, and T. H. Teeri, "Functional characterization and expression of GASCL1 and GASCL2, two anther-specific chalcone synthase like enzymes from Gerbera hybrida", Phytochemistry, vol. 134, pp. 38-45, 2017.
Zhu, L., S. Mattila, R. Matomäki, L. Mollo, S. Ahamed, S. M. Abdou, H. Bashandy, and T. H. Teeri, "Highly Efficient Production of Dihydroflavonol 4-Reductases in Tobacco Cells and Refinement of the BuOH-HCl Enzymatic Assay", Recent Advances in Polyphenol Research, Volume 8, New York, Wiley. online library., 2023.
Bashandy, H., J. S., and T. H. Teeri, "Within leaf variation is the largest source of variation in agroinfiltration of Nicotiana benthamiana", Kasvitieteen Päivät, Turku, Finland, 2016.
Bashandy, H., S. Jalkanen, and T. H. Teeri, "Within leaf variation is the largest source of variation in agroinfiltration of Nicotiana benthamiana.", Plant methods, vol. 11, pp. 47, 2015. Abstract

BACKGROUND: Transient gene expression utilizing syringe agroinfiltration offers a simple and efficient technique for different transgenic applications. Leaves of Nicotiana benthamiana show reliable and high transformation efficiency, but in quantitative assays also a certain degree of variation. We used a nested design in our agroinfiltration experiments to dissect the sources of this variation.

RESULTS: An intron containing firefly luciferase gene was used as a reporter for agroinfiltration. A number of 6 week old tobacco plants were infiltrated for their top leaves, several samples were punched from the leaves after 2 days of transient expression, and protein extracts from the samples were repeatedly measured for luciferase activity. Interestingly, most of the variation was due to differences between the sampling spots in the leaves, the next important source being the different leaves on each plant. Variation between similar experiments, between plants and between repetitive measurements of the extracts could be easily minimized.

CONCLUSIONS: Efforts and expenditure of agroinfiltration experiments can be optimized when sources of variation are known. In summary, infiltrate more plants but less leaves, sample more positions on the leaf but run only few technical replicates.

Bashandy, H., M. Pietiäinen, E. Carvalho, K. - J. Lim, P. Elomaa, S. Martens, and T. H. Teeri, "Anthocyanin biosynthesis in gerbera cultivar 'Estelle' and its acyanic sport 'Ivory'.", Planta, vol. 242, issue 3, pp. 601-11, 2015 Sep. Abstract

MAIN CONCLUSION: Identification of distinct allelic versions for dihydroflavonol 4-reductase in gerbera cultivars reveals that gerbera DFR enzymes have strong substrate preference in vivo that is not reflected to the activity in vitro. Flavonoids in the model ornamental plant Gerbera hybrida consist of flavones, flavonols and anthocyanins. Anthocyanins accumulate in the adaxial epidermis of petals and give the different cultivars their characteristic red and violet colour. Both pelargonidin and cyanidin derivatives are found in gerbera, but none of the cultivars contain delphinidin. 'Ivory', a cultivar with white petals, is a sport of the pelargonidin-containing pink cultivar 'Estelle', i.e. it originates from an acyanic branch of 'Estelle'. In this work, four different alleles encoding dihydroflavonol 4-reductase (DFR) were identified in gerbera cultivars. We found that, in contrast to 'Estelle' with the functional allele GDFR1-2, 'Ivory' carries a mutation in this gene that results in an inactive enzyme. Interestingly, 'Ivory' also expresses a second, nonmutated allele (GDFR1-3) in petal epidermi, leading to extractable DFR activity but not to anthocyanin biosynthesis. The second allele encodes a protein identical in amino acid sequence to the DFR of the cyanidin-containing variety 'President'. Pelargonidin-containing cultivars do not react to the flavonoid 3'-hydroxylase inhibitor tetcyclacis, but cyanidin-containing cultivars lose their colour, instead of starting to synthesise pelargonidins, indicating the specificity of GDFR1-3 for the cyanidin pathway. This explains why petals of 'Ivory' are white, even when it has lost only one of the two enzymatically functional DFR forms, and shows that anthocyanin biosynthesis in gerbera is under more complex regulation than earlier thought.

Bashandy, H., E. Carvalho, S. Martens, and T. H. Teeri, "Flavonoid biosynthetic pathway in Gerbera hybrida", 8th International Workshop on Anthocyanins (IWA), Montpellier, France., 2015.
Bashandy, H., M. Pietiäinen, E. Carvalho, S. Martens, and T. H. Teeri, "Anthocyanin biosynthesis in gerbera cultivars Estelle and Ivory", ICP2014 & 8th Tannin, Japan, 2014.
Cassetta, A., D. Lamba, H. Bashandy, T. Teeri, and S. Martens, "Structural analysis of two distinct dihydroflavonol 4-reductases in Gerbera Hybrids", IWA, Porto, 2013.
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