Details

Autor(en) / Beteiligte

• Lin, Meng-Han
• Gresshoff, Peter M.
• Indrasumunar, Arief
• Ferguson, Brett J.

Titel

pHairyRed： A Novel Binary Vector Containing the DsRed2 Reporter Gene for Visual Selection of Transgenic Hairy Roots

Ist Teil von

• Molecular plant, 2011-05, Vol.4 (3), p.537-545

Ort / Verlag

England: Elsevier Inc

Erscheinungsjahr

2011

Link zum Volltext

Quelle

Free E-Journal (出版社公開部分のみ）

Beschreibungen/Notizen

• We developed a new plant transformation vector, pHairyRed, for enabling high throughput, non-destructive selection of Agrobacterium rhizogenes-mediated ＇hairy-root＇ transformation, pHairyRed allows easy in planta visualiza- tion of transgenic tissue with minimal disturbance to the plant. The DsRed2 reporter gene, encoding a red fluorescent protein, was cloned to yield pHairyRed （harbouring a multiple cloning site）, which was used with the highly efficient K599 A. rhizogenes strain to infect soybean （Glycine max L. Merrill） plants. DsRed2 fluorescence was easily detected in planta for the duration of a 5-week study with negligible levels of background autofluorescence. This enabled visual selection of transformed roots and subsequent excission of non-transformed roots, pHairyRed-transformed roots nodu- lated normally when inoculated with Bradyrhizobiumjaponicum. Within the nodule, DsRed2 fluorescence was plant-spe- cific, being absent in the bacteroid-dominated nodule infected zone. To test the reliability of pHairyRed as a high-fidelity binary vector reporter system, the gene encoding the soybean Nod factor receptor, GmNFRloL, was cloned into the vector for use in a complementation study with a non-nodulating nfrloL mutant of soybean. Complementation was achieved and, without exception, DsRed2 fluorescence was detected in all hairy roots that successfully formed nodules （100%, n = 34）. We anticipate broad application of this reporter system for the further analysis of root-related events in soybean and related legumes.