Arthur K. Weissinger

Scaffold attachment regions increase reporter gene expression in stably transformed plant cells.

The yeast ARS-1 element contains a scaffold attachment region (SAR) that we have previously shown can bind to plant nuclear scaffolds in vitro. To test effects on expression, constructs in which a chimeric beta-glucuronidase (GUS) gene was flanked by this element were delivered into tobacco suspension cells by microprojectile bombardment. In stably transformed cell lines, GUS activity averaged 12-fold higher (24-fold on a gene copy basis) for a construct containing two flanking SARs than for a control construct lacking SARs. Expression levels were not proportional to gene copy number, as would have been predicted if the element simply reduced position effect variation. Instead, the element appeared to reduce an inhibitory effect on expression in certain transformants containing multiple gene copies. The effect on expression appears to require chromosomal integration, because SAR constructs were only twofold more active than the controls in transient assays.

Transgenic tobacco plants and their progeny derived by microprojectile bombardment of tobacco leaves.

Transgenic tobacco plants and progeny carrying coding sequences for neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) were recovered following microprojectile bombardment of tobacco leaves. Transgenic plants were regenerated from bombarded leaf pieces of tobacco cvs. ‘Xanthi’ and ‘Ky 17’ which were cultured in the presence of 100 or 200 μg/ml kanamycin for six to eight weeks. Among 160 putative transgenic plants from at least 16 independent transformation events 76% expressed NPTII, and 50% expressed GUS. Southern analysis of plants expressing either one or both of the enzymes indicated DNA in high molecular weight DNA in 8 of 9 independent transformants analyzed. Two independent transformants and their progeny were analyzed in detail. Analysis of progeny for quantitative enzyme levels of NPTII and GUS, and Southern analysis of parents and progeny clearly demonstrated that the genes were transmitted to progeny. One transformant demonstrated Mendelian ratios for seed germination on kanamycin-containing medium while the other transformant had non-Mendelian ratios. DNA analysis of progeny indicate complex integration of the plasmid DNA, and suggest that rearrangements of this DNA has occurred. These results are consistent with other methods of direct DNA uptake into cells, and verify that the microprojectile bombardment method is capable of DNA delivery into intact plant cells which can give rise to transgenic plants and progeny.

Regeneration of transgenic peanut plants from stably transformed embryogenic callus

Abstract Embryogenic tissue cultures of Arachis hypogaea L. (peanut or groundnut), have been transformed via microprojectile bombardment. We introduced a gene ( hph ) conferring resistance to the antibiotic hygromycin under the control of the CaMV 35S promoter. Selection for resistant callus was initiated 4–5 weeks post-bombardment on medium containing 10–20 mg/l hygromycin. Twelve percent of the bombardments resulted in recovery of a transgenic cell line. An average of two transgenic embryogenic cell lines was isolated per bombardment experiment over 4–6 months of continuous selection. Each bombardment experiment consisted of 11–19 plates, and each plate contained approximately fourteen 25 mm 2 embryogenic callus pieces. Thus, nearly 1% of the bombarded callus pieces produced a stably transformed cell line. Over 100 plants have been regenerated collectively from all of the transformed cell lines. The presence and integration of foreign DNA in hygromycin-resistant callus lines and regenerated plants has been confirmed by polymerase chain reaction amplification of a defined portion of the chimeric gene and by Southern hybridization analysis. Hygromycin resistance was expressed in leaflets from transformed plants which remained green when cultured on basal medium containing hygromycin. Leaflets from control, non-transformed plants turned brown within 3 weeks on the hygromycin-containing medium.

Growth, productivity, and competitiveness of introgressed weedy Brassica rapa hybrids selected for the presence of Bt cry1Ac and gfp transgenes

Concerns exist that transgenic crop × weed hybrid populations will be more vigorous and competitive with crops compared with the parental weed species. Hydroponic, glasshouse, and field experiments were performed to evaluate the effects of introgression of Bacillus thuringiensis (Bt) cry1Ac and green fluorescent protein (GFP) transgenes on hybrid productivity and competitiveness in four experimental Brassica rapa × transgenic Brassica napus hybrid generations (F1, BC1F1, BC2F1 and BC2F2). The average vegetative growth and nitrogen (N) use efficiency of transgenic hybrid generations grown under high N hydroponic conditions were lower than that of the weed parent (Brassica rapa, AA, 2n = 20), but similar to the transgenic crop parent, oilseed rape (Brassica napus, AACC, 2n = 38). No generational differences were detected under low N conditions. In two noncompetitive glasshouse experiments, both transgenic and nontransgenic BC2F2 hybrids had on average less vegetative growth and seed production than B. rapa. In two high intraspecific competition field experiments with varied herbivore pressure, BC2F2 hybrids produced less vegetative dry weight than B. rapa. The competitive ability of transgenic and nontransgenic BC2F2 hybrids against a neighbouring crop species were quantified in competition experiments that assayed wheat (Triticum aestivum) yield reductions under agronomic field conditions. The hybrids were the least competitive with wheat compared with parental Brassica competitors, although differences between transgenic and nontransgenic hybrids varied with location. Hybridization, with or without transgene introgression, resulted in less productive and competitive populations.

Genetic transformation of Norway spruce (Picea abies (L.) Karst) using somatic embryo explants by microprojectile bombardment.

Stable transformation of Norway spruce tissue has been obtained following bombardment of mature somatic embryos with pRT99gus, a plasmid that contains neo coding for NPTII, and gusA, coding for β-glucuronidase, both fused to the CaMV 35S promoter. At least 8 lines have been stably transformed (over 15 months in culture) following bombardment and selection on kanamycin. Polymerase chain reaction analyses showed a high frequency of cotransformation of the gusA and neo genes. The frequency of coexpression of the selected and unselected markers was 100%. DNA/DNA hybridization of one transformed line provided conclusive evidence of stable integration and showed copy numbers of over 10 plasmid sequences per genome. None of the transformed lines has remained embryogenic.

Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop × weed hybrid generations

The level of transgene expression in crop × weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T1 single-locus insert GFP/Bacillus thuringiensis (Bt) transgenic canola (Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC1F1, BC2F1) were produced by backcrossing various GFP/Bt transgenic canola (B. napus, cv Westar) and birdseed rape (Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC2F2 Bulk) were generated by crossing BC2F1 individuals in the presence of a pollinating insect (Musca domestica L.). The ploidy of plants in the BC2F2 Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F1 hybrid generations contained 95–97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15–29% presence in the BC2F2 Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC2F2 Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F1, BC1F1 and BC2F1). These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress. This phenomenon must be considered in the development of risk-management strategies.

Transient expression from microprojectile-mediated DNA transfer in pinus taeda.

SummaryTransfer of plasmid DNA to Pinus taeda L. (loblolly pine) cotyledon cells by microprojectile bombardment has been demonstrated using beta-glucuronidase (GUS). GUS histochemical staining indicated active enzyme in localized centers (blue spots) 24 hours after bombardment. GUS expression declined during subsequent culture, but remained detectable in meristematic tissue 62 days post-bombardment, however, transgenic shoots were not recovered. Localized GUS expression events resulted predominantly from single-cell events containing one microprojectile. The staining pattern was complex, with indigo found both in the central target cell and in adjacent cells. Cellular damage sustained by GUS-positive cells ranged from undetectable to sufficiently extensive to cause cell death. Microprojectile bombardment provides a useful method to assay transient gene expression in loblolly pine and has potential for the production of transgenic plants in pine.

Amplicon-plus Targeting Technology (APTT) for rapid production of a highly unstable vaccine protein in tobacco plants

High-level expression of transgenes is essential for cost-effective production of valuable pharmaceutical proteins in plants. However, transgenic proteins often accumulate in plants at low levels. Low levels of protein accumulation can be caused by many factors including post-transcriptional gene silencing (PTGS) and/or rapid turnover of the transgenic proteins. We have developed an Amplicon-plus Targeting Technology (APTT), by using novel combination of known techniques that appears to overcome both of these factors. By using this technology, we have successfully expressed the highly-labile L1 protein of canine oral papillomavirus (COPV L1) by infecting transgenic tobacco plants expressing a suppressor of post-transcriptional gene silencing (PTGS) with a PVX amplicon carrying a gene encoding L1, and targeting the vaccine protein into the chloroplasts. Further, a scalable “wound-and-agrospray” inoculation method has been developed that will permit high-throughput Agrobacterium inoculation of Nicotiana tabacum, and a spray-only method (named “agrospray”) for use with N. benthamiana to allow large-scale application of this technology. The good yield and short interval from inoculation to harvest characteristic of APTT, combined with the potential for high-throughput achieved by use of the agrospray inoculation protocol, make this system a very promising technology for producing high value recombinant proteins, especially those known to be highly labile, in plants for a wide range of applications including producing vaccines against rapidly evolving pathogens and for the rapid response needed to meet bio-defense emergencies.

Spatial and temporal patterns of green fluorescent protein (GFP) fluorescence during leaf canopy development in transgenic oilseed rape, Brassica napus L.

The green fluorescent protein (GFP) holds promise as a field-level transgene marker. One obstacle to the use of GFP is fluorescence variability observed within leaf canopies. In growth chamber and field experiments, GFP fluorescence in transgenic oilseed rape (Brassica napus) was shown to be variable at each leaf position over time and among different leaves on the same plant. A leaf had its highest GFP fluorescence after emergence and, subsequently, its fluorescence intensity decreased. GFP fluorescence intensity was directly correlated with the concentration of soluble protein. The concentration of the genetically linked recombinant Bacillus thuringiensis (Bt) cry1Ac endotoxin protein also was examined, and GFP fluorescence was positively correlated with Bt throughout development. The results show that GFP can be used as an accurate transgene marker but that aspects of plant developmental should be taken into account when interpreting fluorescence measurements.

Hypoallergenic legume crops and food allergy: factors affecting feasibility and risk.

Currently, the sole strategy for managing food hypersensitivity involves strict avoidance of the trigger. Several alternate strategies for the treatment of food allergies are currently under study. Also being explored is the process of eliminating allergenic proteins from crop plants. Legumes are a rich source of protein and are an essential component of the human diet. Unfortunately, legumes, including soybean and peanut, are also common sources of food allergens. Four protein families and superfamilies account for the majority of legume allergens, which include storage proteins of seeds (cupins and prolamins), profilins, and the larger group of pathogenesis-related proteins. Two strategies have been used to produce hypoallergenic legume crops: (1) germplasm lines are screened for the absence or reduced content of specific allergenic proteins and (2) genetic transformation is used to silence native genes encoding allergenic proteins. Both approaches have been successful in producing cultivars of soybeans and peanuts with reduced allergenic proteins. However, it is unknown whether the cultivars are actually hypoallergenic to those with sensitivity. This review describes efforts to produce hypoallergenic cultivars of soybean and peanut and discusses the challenges that need to be overcome before such products could be available in the marketplace.