| Abstract | A robust, reproducible method of Agrobacterium-mediated transformation was developed for Lupinus mutabilis Sweet (tarwi), a large-seeded Andean legume. Initially, a regeneration and transformation protocol was developed using a plasmid which contained a bifunctional fusion gene conferring both β-glucuronidase (gus) and neomycin phosphotransferase activities, under the control of a constitutive 35S35SAMV promoter. The tissue explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. Using a series of tissue culture media for cocultivation, shoot initiation, shoot elongation, and rooting, kanamycin-resistant transgenic plants were recovered from approximately 1% of the explants. This transformation protocol was further used with a construct that contained the human adenosine deaminase (hADA) gene under the control of a legumin seed-specific promoter, also with a kanamycin resistance cassette for chemical selection. Changes made during the course of this study, which included adjustments to the antibiotic concentration during the shoot elongation and rooting phases plus the incorporation of techniques to improve ventilation in the tissue culture system, resulted in major improvements in shoot quality and, most significantly, rooting. The outcome was an increased frequency of transgenic plant recovery (7.4%), with a low (9.6%) rate of plants that escaped selection. The inheritance of the hADA gene was documented and showed the expected Mendelian segregation pattern. The produced hADA protein was a fully functional enzyme and localized only in the seed, as expected. Thus, this legume species is an excellent candidate for a nonfood plant host platform for the production of plant-made proteins. |
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