Poster: Emerging technologies
Abs #
906: Engineering the carrot chloroplast genome via somatic embryogenesis to confer salt tolerance and facilitate transgene containment
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Presenter: |
Kumar, Shashi , skumar@mail.ucf.edu |
Authors | Kumar, Shashi (A) Amit, Dhingra (A) Henry, Daniell (A) | | Affiliations: |
(A): University of Central Florida
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Transgenic carrot plants exhibiting high levels of salt tolerance were rapidly regenerated from carrot cell cultures bombarded with carrot specific chloroplast vectors via somatic embryogenesis. Carrot calli, leaf, and stem segments were bombarded with carrot species-specific plastid vectors and incubated for 2 days in the dark. After 2 days different cultures were selected on MSB medium containing different concentrations of betaine aldehyde and spectinomycin. Transgenic calli obtained from different tissues of carrot cultures were tested for site-specific integration of transgenes into plastid genomes by PCR and Southern blot analyses. Transformed embryogenic cultures were maintained in vitro or converted into plants that transferred to soil in pots to induce the mature taproot system. Maximum transformation efficiency (13.3%) was observed in carrot cell cultures when compared to cotyledons (10.0%) and stem segments (6.25%). Transgenic calli derived from cultured cells, cotyledons and stem segments expressing badh transgene were green in color whereas non-transgenic cells were yellow in color. High BADH enzyme activity and protein accumulation was observed in carrot taproot (74.8% of leaves). Transformed carrot cells were able to sustain and proliferate in the liquid medium containing NaCl than untransformed cells (8.75 g transgenic cells compared to 1.29 g in untransformed control at 100 mM NaCl). Transgenic plants carrying the badh transgene grew in NaCl (up to 400 mM) whereas untransformed plants exhibited severe growth retardation in 200 mM or higher concentrations of NaCl. This is the first plastid transformation achieved using non-green explants via somatic embryogenesis and should facilitate oral delivery of therapeutic proteins.
