Location: Soybean and Nitrogen Fixation Research
Title: Non-antibiotic selection systems for soybean somatic embryos: the lysine analog aminoethyl-cysteine as a selection agent Authors
|Suryadevara, S. - UNIV OF KENTUCKY|
|Mamadou, Lewamy - UNIV OF KENTUCKY|
|Mcconnell, Matt - UNIV OF KENTUCKY|
|Raghuveer, Polisetty - UNIV OF KENTUCKY|
|Hildebrand, David - UNIV OF KENTUCKLY|
Submitted to: BioMed Central (BMC)Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 9, 2009
Publication Date: N/A
Interpretive Summary: Although hygromycin is normally used for the selection, it is an antibiotic and not an ideal for the final product. Several agents including lysine analog, aminoethyl-cysteine (AEC), and other commercially available non-antibiotic agents were used for selection of soybean somatic embryonic transformation. The lysine analog AEC was successfully used at a 5 mM concentration. Only transgenic soybeans were able to grow at this concentration. AEC was preferable since it is a natural compound and proven to be very tight with no false positive transformation.
Technical Abstract: In soybean somatic embryo transformation, the standard selection agent currently used is hygromycin. However, hygromycin being an antibiotic is not ideal in the final product. When tested against different alternate selection agents our studies show that 0.16 µg/ mL glufosinate, 40 mg/L isopropylamine-glyphosate, 0.5 mg/mL (S-(2 aminoethyl)-L-cysteine) (AEC) and the acetolactate synthase (ALS) inhibitors Exceed® and Synchrony® both at 150 µg/mL can be used as selectable markers for soybean somatic embryos. Even at the concentration of 2 mg/mL, lysine+threonine (LT) were poor selection agents. The use of AEC may be preferable since it is a natural compound. Unlike plant enzymes dihydrodipicolinate synthase (DHPS) E. coli is not feed-back inhibited by physiological concentrations of lysine. The dapA gene which codes for E. coli DHPS was expressed in soybean somatic embryos under the CaMV 35S promoter using biolistics transformation. The lysine analogue AEC was used as selection agent at a 5 mM concentration to select transgenic embryos. AEC selection for soybean turned out to be very tight with no false positives in the transformants. Only transgenic soybeans were able to grow at this concentration of AEC; no escapes were observed. AEC insensitive dapA genes + moderate concentrations of AEC can be used to effectively select transgenic soybean somatic embryos without the need of antibiotic resistance genes.