Field Assessment of Outcrossing from Transgenic Pea (Pisum Sativum L.) Plants
The frequency of outcrossing from a transgenic line of peas into three cultivars ('Carneval', 'Montana', 'Tipu') was studied in the field in 1997 and 1999. Two dominant traits, normal leaf form and a highly-expressed β-glucuronidase (gusA) gene, were used as markers of pollen transfer. Because of heterogeneity in the commercial seed sources, leaf form alone was unreliable for assessing pollen migration into 'trap' plants. Of approximately 9000 offspring tested, only five plants scored positive for the presence of both markers. All five were located in 'trap' plots situated near the transgenic line. This represents a mean outcrossing rate of 0.07%.
Agrobacterium-mediated transformation of tarwi (Lupinus mutabilis Sweet), a potential platform for the production of plant-made proteins
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.
Digestion in larvae of Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) with emphasis on α-amylases and oligosaccharidases
Abstract Determinations of carbohydrases, aminopeptidases and acid phosphatase in the larval midgut cells and in the luminal contents of Callosobruchus maculatus and Zabrotes subfasciatus have been carried out. The results showed that larvae of both species displayed similar distribution of digestive enzymes in the intestinal compartments. Most larval digestive enzyme activities were found in the luminal contents. Of the activities found in the midgut tissue, only aminopeptidase is predominant in the membrane fraction. Comparisons of activities recovered from a seed flour mass equivalent to the midgut mass showed that a high percentage of the luminal aminopeptidase activity and, to a lesser extent, α-galactosidase activity, can be derived from the seeds, whereas the other enzymes are produced by the insects. Activities against starch, maltose and maltodextrins were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on differences in elution profiles on hydrophobic chromatography and banding patterns in mildly denaturing electrophoresis, both species showed a multiplicity of glycosidases. The data suggest that the majority of carbohydrate digestion occurs in the midgut lumen, whereas protein digestion should take place partly in the lumen and partly at the cell surface. Larvae of Z. subfasciatus can modulate the levels of α-amylases and α-glucosidase in response to different diets. The complex of carbohydrases found is qualitatively appropriate to digest the free oligosaccharides and oligomaltodextrins produced by α-amylases from the starch granules of host seeds.