Poster: Emerging technologies
Abs #
905: Engineering the cotton chloroplast genome and maternal inheritance of transgenes
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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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Cotton is the single most important textile fiber in the world, grown in more than 90 countries. The first method for cotton chloroplast genetic engineering through somatic embryogenesis is presented here. Cotton plastid transformation was extensively tried using the aadA gene containing species-specific cotton chloroplast vectors. However, no transgenic calli or plants were recovered using spectinomycin as the selection agent due to its lethal effect on cotton cultures. Subsequently, cotton callus cultures were bombarded with the chloroplast specific vector pDD-Gh-aphA6/nptII and transgenic cell lines were selected on medium supplemented with kanamycin. Transformed callus cultures were then multiplied on higher concentrations of kanamycin in order to increase the number of transgenic chloroplasts in cotton cultures. The maximum transformation efficiency (41.9%) was observed when cell cultures were bombarded at 9 cm distance with 650 psi rupture disc, that suggests that higher cell death or production of phenolic compounds at sites of injury may be important determinants in transformation efficiency. Transgenic somatic embryos were matured and elongated into plantlets and transferred to growth chambers for flowering and setting seeds. Stable transgene integration into cotton chloroplast genomes was confirmed by PCR and southern blot analyses. A large number of crosses were made between non-transgenic cotton and chloroplast transgenic. No germination of seeds from F1 crosses (non-transgenic female x male transgenic chloroplast) was observed on the kanamycin selection medium, where as all self-pollinated transgenic chloroplast plants germinated on kanamycin medium confirming maternal inheritance, lack of pollen transmission and Mendelian segregation of transgenes.
