Wheat Transformation Using Cyanamide as a New Selective Agent

Abstract

There is a general need for additional selectable marker genes for plant transformation. Only a few have been reported in wheat (Triticum aestivum L.) transformation experiments, some of which are under patent restriction or have other disadvantages. A new selectable marker gene was identified which can be used to select resistant callus in tissue culture and regenerate transgenic wheat plants. A gene from the soil fungus Myrothecium verrucaria (Albertini & Schwein.) Ditmar:Fr., coding for the enzyme cyanamide hydratase which converts cyanamide into urea, was previously described. In our wheat transformation experiments, the gene conferred resistance to cyanamide at a tissue culture level and therefore cyanamide could be used to select for transformants. At the whole plant level, progeny of transformed wheat plants showed resistance to cyanamide, whereas sensitive plants expressed a lethal necrosis and yellowing when cyanamide was applied. This gene has several potential advantages when compared with other selectable marker genes. Transformed wheat plants can be selected at the tissue culture level and may be able to convert cyanamide into a useful nitrogen compound (fertilizer). The selectable marker gene could be introduced with other genes for value‐added traits in wheat and might also be applicable in other transformation systems.

Two Duplicated Genes DDI2 and DDI3 in Budding Yeast Encode a Cyanamide Hydratase and Are Induced by Cyanamide

“…The cyanamide concentration was monitored by using a colorimetric assay as described (29). Briefly, 100 l of reaction mix was added to 500 l of PBS, followed by adding 400 l of 0.1 M sodium carbonate buffer, pH 10.4, and 200 l of 4% sodium pentacyanoammine ferroate(II) (TCI, S0050) as the color reagent.…”

Genetic transformation of major cereal crops

“…Cyanamide hydratase is an enzyme which converts cyanamide into urea, hence promotes plant growth (Maier-Greiner et al, 1991). By using a cyanamide hydratase-coding gene isolated from the soil fungus Myrothecium verrucaria as selective marker, Weeks et al, (2000) successfully obtained two stable transgenic events, but the TF was only 0.2%. The pmi gene was also investigated as selectable marker gene and the TF could reach 20% in spring wheat UC703 by bombardment .…”

Expression of a fungal cyanamide hydratase in transgenic soybean detoxifies cyanamide in tissue culture and in planta to provide cyanamide resistance

“…1). Based on this capability, Cah has been used as a selectable marker for transformation of Arabidopsis, potato, rice and tomato (Damm, 1998) as well as wheat (Weeks et al, 2000) using cyanamide as the selective agent. Plants apparently do not contain a homologous Cah-like gene, because blast searches of the plant databases did not identify any sequences similar to the Cah gene at either the nucleotide or amino acid sequence level.…”