Transgenic potatoes were engineered to synthesize a cholera toxin B subunit (CTB) pentamer with affinity for GMI-ganglioside. Both serum and intestinal CTB-specific antibodies were induced in orally immunized mice. Mucosal antibody titers declined gradually after the last immunization but were restored following an oral booster of transgenic potato. The cytopathic effect of cholera holotoxin (CT) on Vero cells was neutralized by serum from mice immunized with transgenic potato tissues. Following intraileal injection with CT, the plant-immunized mice showed up to a 60% reduction in diarrheal fluid accumulation in the small intestine. Protection against CT was based on inhibition of enterotoxin binding to the cell-surface receptor GMI-ganglioside. These results demonstrate the ability of transgenic food plants to generate protective immunity in mice against a bacterial enterotoxin.

Oral administration of disease-specific autoantigens can prevent or delay the onset of autoimmune disease symptoms. We have generated transgenic potato plants that synthesize human insulin, a major insulin-dependent diabetes mellitus autoantigen, at levels up to 0.05% of total soluble protein. To direct delivery of plant-synthesized insulin to the gut-associated lymphoid tissues, insulin was linked to the C-terminus of the cholera toxin B subunit (CTB). Transgenic potato tubers produced 0.1% of total soluble protein as the pentameric CTB-insulin fusion, which retained GM1-ganglioside binding affinity and native antigenicity of both CTB and insulin. Nonobese diabetic mice fed transformed potato tuber tissues containing microgram amounts of the CTB-insulin fusion protein showed a substantial reduction in pancreatic islet inflammation (insulitis), and a delay in the progression of clinical diabetes. Feeding transgenic potato tissues producing insulin or CTB protein alone did not provide a significant reduction in insulitis or diabetic symptoms. The experimental results indicate that food plants are feasible production and delivery systems for immunotolerization against this T cell-mediated autoimmune disease.

This article described the population structure of trembling aspen (Populus tremuloides Michx.) in Alberta, a dioecious tree with continuous and wide distribution, and with a primary mode of reproduction through suckering. We studied random amplified polymorphic DNA (RAPD) variation in 249 trees from eight natural populations. Trees within a population were a minimum of 200 m apart to decrease the risk of sampling ramets of a single ortet. Of a total of 28 amplified RAPD products (bands) from five random oligonucleotide primers, the frequencies at seven (25%) were heterogeneous across populations and the percentage of polymorphism averaged 90.2 per population. Estimates of Shannon's phenotypic diversity index ranged between 0.58 and 0.69 among populations, averaging 0.65. There were 246 multiband phenotypes among the 249 trees; three were each shared by two trees from different populations and the remaining 243 were unique. Thus, trees within populations probably were different clones. Analysis of molecular variance partitioned the RAPD variation into the among- and within-population components. The within-population component accounted for 97.4% of the variation and was significantly different from zero at the 2% level of probability. The among-population component, although accounting for only 2.6% of the variation, was significantly different from zero at the 1% level of probability. Pairwise tests for the homogeneity of the RAPD variance between populations suggested significant divergences among 18 of the 28 (64%) population pairs.

A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (M(r) approximately 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (M(r) approximately 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world.

In the present study, a total of 142 trees sampled from five populations of trembling aspen (Populus tremuloides Michx.) in Alberta was analyzed by the polymerase chain reaction (PCR) with five random oligonucleotide primers. Null-allele frequencies of 28 putative RAPD loci were estimated using the given departure from Hardy-Weinberg equilibrium (F1S) previously estimated with isozyme markers for the same population. Nucleotide divergence between populations was then estimated in a fashion similar to restriction-fragment data, but considering the dominance of the RAPDs. The average of nucleotide divergence between populations was in the order of 0.0005 and nucleotide divergence were found to be highly correlated with geographic distance. The results suggest that isolation by distance may be an important factor in the genetic differentiation of trembling aspen.