| Using the model plant species, Aloe (Aloe barbadensis), a monocot, and sunflower (Helianthus annuus), a dicot, new methods for plant tissue culture and Agrobacterium transformation were developed and combined to advance the field of agricultural biotechnology and support the development of pharmaceuticals from genetically engineered plants. Among the new methods, which were developed, are: (1) a new method for the transformation of virulent strains of Agrobacterium with binary plasmids, (2) a method for the production of embryogenic calli from Aloe tissue explants, which can be used for Agrobacterium-mediated transformation of Aloe and as a method for mass propagation of Aloe, (3) a method for transforming Aloe with virulent Agrobacterium strains and, (4) methods to produce and culture a kanamycin resistant sunflower (Helianthus annuus) hairy root cell line. The new method for introducing binary plasmids into virulent strains of Agrobacterium is a convenient and efficient alternative to existing methods and insures the virulence of the Agrobacterium by using only low temperature incubation steps. Combining the method developed for Agrobacterium and the methods for transformation and mass propagation of Aloe, it may be possible to produce a transgenic Aloe cell line and mass propagate the transgenic line via somatic embryogenesis. Combining the method developed for Agrobacterium with the method for transforming sunflower tissue with Agrobacterium rhizogenes and the method for culturing sunflower hairy roots, it was possible to produce and propagate a very fast growing transgenic sunflower hairy root cell line in plant cell culture using a specialized high nitrogen medium. Sunflower hairy root cell lines produced using these techniques can be grown in a liquid bioreactor for the production of plant secondary metabolites or products of foreign gene expression, including transgenic peptide pharmaceuticals. |
