Non-antibiotic selection systems for soybean somatic embryos: the lysine analog aminoethyl-cysteine as a selection agent.
BACKGROUND
In soybean somatic embryo transformation, the standard selection agent currently used is hygromycin. It may be preferable to avoid use of antibiotic resistance genes in foods. The objective of these experiments was to develop a selection system for producing transgenic soybean somatic embryos without the use of antibiotics such as hygromycin.


RESULTS
When tested against different alternate selection agents our studies show that 0.16 microg/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 microg/mL inhibited soybean somatic embryo growth. 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 the plant enzyme, dihydrodipicolinate synthase (DHPS) from 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 control of the CaMV 35S promoter. Following introduction of the construct into embryogenic tissue of soybean, transgenic events were recovered by incubating the tissue in liquid medium containing AEC at a concentration of 5 mM. Only transgenic soybeans were able to grow at this concentration of AEC; no escapes were observed.


CONCLUSIONS
Genetically engineered soybeans expressing a lysine insensitive DHPS gene can be selected with the non-antibiotic selection agent AEC. We also report here the inhibitory effects of glufosinate, (isopropylamine-glyphosate) (Roundup), AEC and the ALS inhibitors Exceed and Synchrony against different tissues of soybean.
Rao
Suryadevara S
SS
Plant & Soil Sciences, University of Kentucky, Lexington, KY 40546, USA. ssrao1@uky.edu
Mamadou
Lewamy
L
McConnell
Matt
M
Polisetty
Raghuveer
R
Kwanyuen
Prachuab
P
Hildebrand
David
D
D003470:Culture Media; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D018744:DNA, Plant / Q000032:analysis; D005818:Genetic Engineering / Q000379:methods*; D006540:Herbicides / Q000494:pharmacology; D006836:Hydro-Lyases / Q000235:genetics*; D008239:Lysine / Q000031:analogs & derivatives*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000254:growth & development; D013025:Glycine max / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development
D003470:Culture Media; D018744:DNA, Plant; D006540:Herbicides; C000204:S-2-aminoethyl cysteine; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase; D008239:Lysine; D003545:Cysteine