Non-antibiotic selection systems for soybean somatic embryos: the lysine analog aminoethyl-cysteine as a selection agent
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 if intended for commercial purposes.
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 ug/ mL glufosinate, 40 mg/L isopropylamine-glyphosate, 0.5 mg/mL (S-(2 aminoethyl)-L-cysteine) (AEC) and the acetolactate synthase (ALS) inhibitors Exceed(R) and Synchrony(R) both at 150 ug/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 escapeswere 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(R)), AEC and the ALS inhibitors Exceed(R) and Synchrony(R) against different tissues of soybean.
Author: Suryadevara RaoLewamy MamadouMatt McConnellRaghuveer PolisettyPrachuab KwanyuenDavid Hildebrand
Credits/Source: BMC Biotechnology 2009, 9:94
Published on: 2009-11-18
Copyright by the authors listed above - made available via BioMedCentral (Open Access). Please
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