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 x 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 x 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.     

Persistence of transgenes in wild rice populations depends on the interaction between genetic background of recipients and environmental conditions

The persistence of transgenes in wild populations may cause unintended ecological consequences, and the possibility of transgenes' persistence and introgression is dependent on fitness performance of transgenic crop–wild hybrids. To investigate the effects of transgene and genotype × environment on the fitness of crop–wild rice hybrids, a total of 11 cross‐combination progenies between insect‐resistant transgene (CpTI and Bt/CpTI) rice lines and wild rice (Oryza rufipogon) were evaluated at different sites with contrasting insect treatments. The results showed that fitness performance varied between transgenic hybrids having different wild parents and under different environmental conditions, indicating that fitness effects of transgenes on hybrid progenies depend heavily on the genetic background of recipient plants and growing environment. Significant fitness advantages conferred by transgenes were found only in some hybrids under high insect pressure condition, demonstrating that the level of target insects in the field environment influences the persistence and spread of insect‐resistant transgenes in wild rice populations. These findings suggest that evolutionary fate of escaped transgenes is different in wild populations with diverse genetic backgrounds under various environmental conditions.     

No effect of transgene and strong wild parent effects on seed dormancy in crop–wild hybrids of rice: implications for transgene persistence in wild populations

Soil seed banks act as a gene pool for local plant species and, as such, can buffer local populations, especially those experiencing challenging environmental conditions. Seed dormancy has important implications to dynamics of soil seed banks. Therefore, estimating the seed dormancy of transgenic crop–wild hybrids could shed light on the persistence of transgenes in wild‐plant soil seed banks. Individuals from eight populations of wild rice Oryza rufipogon were crossed with those of three insect‐resistant transgenic rice lines. Selfed (F2–F4) and backcrossed populations (BC1, BC1F2 and BC1F3) were then made from the hybrids. Seed germination was tested under three treatments: (a) normal; (b) overwintering in soil; and (c) one‐week heat‐shocking. The effects of transgene, wild parent and hybrid generation on hybrid seed germination were examined. No significant effect of insect‐resistant transgenes (Bt and CpTI) was detected on the seed dormancy of crop–wild hybrids, while a significant wild parent effect was found. The seeds of advanced generation hybrids have higher germination percentages and lower dormancy than do those of F1 and BC1 generations. The study showed that the dormancy of hybrid seeds was determined mainly by their genetic backgrounds. All hybrid seeds have higher germination percentages and lower dormancy (and, consequently, a poorer overwintering ability), compared with wild seeds, and reduce dormancy would contribute to a fitness disadvantage, compared with wild types. Therefore, such seeds might form part of naturally occurring soil seed banks, through which crop genes would persist in wild populations.     

Genetic transformation of Rangpur lime (Citrus limonia osbeck) with thebO (bacterio-opsin) genen and its initial evaluation forPhytophthora nicotianae resistance

Transgenic plants expressing the bacterio-opsin (bO) gene can spontaneously activate programmed cell death (ped) and may enhance broad-spectrum pathogen resistance by activating an intrinsic defense pathway in plant species such as tobacco and potato. In this work, we produced transgenic Rangpur lime plants with thebO gene, viaAgrobacterium tumefaciens-mediated transformation, and evaluated these plants forPhytophthora nicotianae resistance. Two transgenic lines were successfully regenerated and transformation was confirmed by GUS activity assay, PCR analysis, Southern, Northern and Western blot analyses, in addition to detecting the expressed bO protein by an immunological approach. Evaluation forPhytophthora nicotianae resistance was carried out by plant inoculations with the pathogen and quantification of the affected area. One of the two transgenic lines showed greater tolerance to the fungal pathogen as compared to the control, with significantly smaller stem lesions after pathogen challenge. This increase in pathogen tolerance is correlated with a significantly higher level of transgene expression in this line when compared with the other transgenic line. This is the first report of the introduction of a potentially important gene into Rangpur lime to provide novel pathogen tolerance.     

Effects of genomic position and copy number of Acyl-ACP thioesterase transgenes on the level of the target fatty acids in Brassica napus L.

The effects of genomic position and copy number of acyl-acyl carrier protein (ACP) thioesterase (TE) transgenes on the major target fatty acid, either lauric acid (C12:0) or palmitic acid (C16:0) depending on the TE, in transgenic Brassica napus seed oil were investigated. Four transgenic parental lines, transformed individually with the bay-TE (Uc FatB1), elm-TE (Ua FatB1), nutmeg-TE (Mf FatB1) and Cuphea-TE (Ch FatB1) transgenes, were crossed with the non-transgenic recipient genotypes '212/86' or 'QO4'. Bay-TE and Cuphea-TE F₁ seeds, which carry half the number of the construct copies compared to the self-pollinated seeds of the transgenic parents, showed significantly lower levels of the target fatty acid. Doubled haploid (DH) lines were developed through microspore culture from F₁ hybrids with the elm-TE or the Cuphea-TE transgenes. DH lines carrying one to five copies of the Cuphea-TE transgene displayed a positive correlation between transgene copy number and the target fatty acid C16:0 level (r = 0.77**). DH lines with elm-TE transgene copies at four different loci showed different C16:0 levels, with one of the loci (E-II) leading to significantly higher C16:0 levels. This study supports the importance of the selection of high transgene copy number and/or the optimum genomic integration site in order to achieve maximum expression levels of the target fatty acid in transgenic oil quality modification.     

Limited fitness advantages of crop-weed hybrid progeny containing insect-resistant transgenes (Bt/CpTI) in transgenic rice field

Background

The spread of insect-resistance transgenes from genetically engineered (GE) rice to its coexisting weedy rice (O. sativa f. spontanea) populations via gene flow creates a major concern for commercial GE rice cultivation. Transgene flow to weedy rice seems unavoidable. Therefore, characterization of potential fitness effect brought by the transgenes is essential to assess environmental consequences caused by crop-weed transgene flow.

Methodology/Principal Findings

Field performance of fitness-related traits was assessed in advanced hybrid progeny of F₄ generation derived from a cross between an insect-resistant transgenic (Bt/CpTI) rice line and a weedy strain. The performance of transgene-positive hybrid progeny was compared with the transgene-negative progeny and weedy parent in pure and mixed planting of transgenic and nontransgenic plants under environmental conditions with natural vs. low insect pressure. Results showed that under natural insect pressure the insect-resistant transgenes could effectively suppress target insects and bring significantly increased fitness to transgenic plants in pure planting, compared with nontransgenic plants (including weedy parent). In contrast, no significant differences in fitness were detected under low insect pressure. However, such increase in fitness was not detected in the mixed planting of transgenic and nontransgenic plants due to significantly reduced insect pressure.

Conclusions/Significance

Insect-resistance transgenes may have limited fitness advantages to hybrid progeny resulted from crop-weed transgene flow owning to the significantly reduced ambient target insect pressure when an insect-resistant GE crop is grown. Given that the extensive cultivation of an insect-resistant GE crop will ultimately reduce the target insect pressure, the rapid spread of insect-resistance transgenes in weedy populations in commercial GE crop fields may be not likely to happen.     

Engineered selective plant male sterility through pollen‐specific expression of the EcoRI restriction endonuclease

Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen‐specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible‐to‐no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand‐crossed to both male‐sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000–40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male‐sterile tobacco, and 900–2100 seeds per male‐sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI‐driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.     

Transgenic American elm shows reduced Dutch elm disease symptoms and normal mycorrhizal colonization

The American elm (Ulmus americana L.) was once one of the most common urban trees in eastern North America until Dutch-elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, eliminated most of the mature trees. To enhance DED resistance, Agrobacterium was used to transform American elm with a transgene encoding the synthetic antimicrobial peptide ESF39A, driven by a vascular promoter from American chestnut. Four unique, single-copy transgenic lines were produced and regenerated into whole plants. These lines showed less wilting and significantly less sapwood staining than non-transformed controls after O. novo-ulmi inoculation. Preliminary observations indicated that mycorrhizal colonization was not significantly different between transgenic and wild-type trees. Although the trees tested were too young to ensure stable resistance was achieved, these results indicate that transgenes encoding antimicrobial peptides reduce DED symptoms and therefore hold promise for enhancing pathogen resistance in American elm.     

Constitutive expression of an inducible lipoxygenase in transgenic tobacco decreases susceptibility to Phytophthora parasitica var. nicotianae

Plant lipoxygenases (LOXs) are key enzymes involved in the generation of fatty acid derivatives, called oxylipins. In tobacco, LOX gene expression and activity are very low in healthy tissues and are highly enhanced in response to infection by Phytophthora parasitica nicotianae and to elicitor treatment. We previously showed, using antisense-LOX1 plants, that expression of the tobacco LOX1 gene is required for the race-cultivar specific resistance of tobacco to Phytophthora parasitica nicotianae. In order to investigate the effect of over-expressing a LOX gene on plant resistance, we transformed tobacco plants with the LOX1 coding sequence fused to the CaMV 35S promoter. Four transgenic lines with enhanced levels of LOX protein and specific activity over control plants were selected for further analysis. These plants were macroscopically indistinguishable from WT plants. Upon stem inoculation, the sense-LOX1 plants displayed a significantly decreased susceptibility to virulent races of Phytophthora parasitica nicotianae, stem lesions being 2- to 3-fold shorter in the transgenic lines than in WT plants. Using a root inoculation assay, the survival rate of sense-LOX1 seedlings was increased about 4-fold compared to their WT counterparts, with 60 to 80% of transgenic plants vs 15 to 20% of WT controls remaining healthy following inoculation with Phytophthora parasitica nicotianae. This is the first demonstration that the over-expression of a LOX gene is sufficient to reduce the susceptibility of a host plant to an oomycete pathogen.     

Field Performance of Transgenic indica Hybrid Rice with Improved Cooking and Eating Quality by Down-regulation of Wx Gene Expression

High amylose content (AC) in rice endosperm is correlated with poor grain quality, particularly in indica hybrid rice. We have generated several homozygous transgenic parent lines of indica hybrid rice carrying an antisense Waxy (Wx) gene and demonstrated that the AC in seeds of these lines decreased dramatically. Two transgenic maintainer lines (L25B and L18B), derived from one of the key maintainer parents of an indica hybrid rice in China, Long-te-fu B (LTF-B), were selected and the antisense Wx gene was subsequently introgressed into the male-sterile counterpart, LTF-A, with the aim to generate improved indica hybrids. The indica hybrids derived from the selected transgenic male-sterile lines and restorer lines were tested for quality and agronomic performance under normal field conditions. Our results demonstrated that the reduction of AC in the homozygous transgenic maintainer lines stably passed down in five successive generations and the improved quality was also found in their relevant transgenic hybrids produced. The other two key characters of rice cooking and eating quality, the gel consistence (GC) and gelatinization temperature (GT), were also improved in the grains of both the transgenic maintainer lines and their relevant hybrids. In addition, no change was observed for most of the agronomic characters of the transgenic maintainer lines and the relevant transgenic hybrids. Although the grain weight of the transgenic line was reduced, the grain yield of the homozygous transgenic parent lines and the transgenic hybrids was similar when compared with that of the wild-type controls. These results suggest that transgenic approaches are an effective way to obtain rice lines with both improved qualities and high yield, especially for indica hybrid rice.     

Characterization of directly transformed weedy <Emphasis Type="Italic">Brassica rapa</Emphasis> and introgressed <Emphasis Type="Italic">B. rapa</Emphasis> with Bt <Emphasis Type="Italic">cry1Ac</Emphasis> and <Emphasis Type="Italic">gfp</Emphasis> genes

Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa × B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.     

Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.)

Molecular analysis of the transgenes bar and gus was carried out over successive generations in six independent transgenic lines of wheat, until the plants attained homozygosity. Data on expression and integration of the transgenes is presented. Five of the lines were found to be stably transformed, duly transferring the transgenes to the next generation. The copy number of the transgenes varied from one to five in the different lines. One line was unstable, first losing expression of and then eliminating both the transgenes in R3 plants. Although the gus gene was detected in all the lines, GUS expression had been lost in R2 plants of all but one line. Rearrangement of transgene sequences was observed, but it had no effect on gene expression. All the stable lines were found to segregate for transgene activity in a Mendelian fashion.     

Transgenic rice as a system to study the stability of transgene expression: multiple heterologous transgenes show similar behaviour in diverse genetic backgrounds

The success of contemporary breeding programmes involving genetic engineering depends on the stability of transgene expression over many generations. We studied the stability of transgene expression in 40 independent rice plant lines representing 11 diverse cultivated varieties. Each line contained three or four different transgenes delivered by particle bombardment, either by cotransformation or in the form of a cointegrate vector. Approximately 75% of the lines (29/40) demonstrated Mendelian inheritance of all transgenes, suggesting integration at a single locus. We found that levels of transgene expression varied among different lines, but primary transformants showing high-level expression of the gna, gusA, hpt and bar transgenes faithfully transmitted these traits to progeny. Furthermore, we found that cry1Ac and cry2A transgene expression was stably inherited when primary transformants showed moderate or low-level expression. Our results show that six transgenes (three markers and three insect-resistance genes) were stably expressed over four generations of transgenic rice plants. We showed that transgene expression was stable in lines of all the rice genotypes we analysed. Our data represent a step forward in the transfer of rice genetic engineering technology from model varieties to elite breeding lines grown in different parts of the world. Received: 22 March 1999 / Accepted: 6 December 1999     

Expression of a transgene exchanged by the recombinase-mediated cassette exchange (RMCE) method in plants

We developed a site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated cassette exchange (RMCE). We produced site-specific transgenic tobacco plants from four target lines and examined expression of the transgene in T1 site-specific transgenic tobacco plants, which were obtained by backcrossing. We found that site-specific transgenic plants from the same target lines showed approximately the same level of expression of the transgene. Moreover, we demonstrated that site-specific transgenic plants showed much less variability of transgene expression than random-integration transgenic plants. Interestingly, transgenes in the same direction at the same target locus showed the same level of activity, but transgenes in different directions showed different levels of activity. The expression levels of transgene did not correlate with those of the target gene. Our results showed that the SDI system could benefit the precise comparisons between different gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic plants.     

Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions.     

Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment

Zea mays transformants produced by particle bombardment of embryogenic suspension culture cells of the genotype A188 × B73 and selected on kanamycin or bialaphos were characterized with respect to transgene integration, expression, and inheritance. Selection on bialaphos, mediated by thebar orpat genes, was more efficient than selection on kanamycin, mediated by thenptII gene. Most transformants contained multicopy, single locus, transgene insertion events. A transgene expression cassette was more likely to be rearranged if expression of that gene was not selected for during callus growth. Not all plants regenerated from calli representing single transformation events expressed the transgenes, and a non-selectable gene (uidA) was expressed in fewer plants than was the selectable transgene. Mendelian inheritance of transgenes consistent with transgene insertion at a single locus was observed for approximately two thirds of the transformants assessed. Transgene expression was typically, but not always, predictable in progeny plants-transgene silencing, as well as poor transgene transmission to progeny, was observed in some plant lines in which the parent plants had expressed the transgene.