Herbicide Resistance Transgene Research Articles

Impact of Intron and Retransformation on Transgene Expression in Leaf and Fruit Tissues of Field-Grown Pear Trees.

Stable and high expression of introduced genes is a prerequisite for using transgenic trees. Transgene stacking enables combining several valuable traits, but repeated transformation increases the risk of unintended effects. This work studied the stability and intron-mediated enhancement of uidA gene expression in leaves and different anatomical parts of pear fruits during field trials over 14 years. The stability of reporter and herbicide resistance transgenes in retransformed pear plants, as well as possible unintended effects using high-throughput phenotyping tools, were also investigated. The activity of β-glucuronidase (GUS) varied depending on the year, but silencing did not occur. The uidA gene was expressed to a maximum in seeds, slightly less in the peel and peduncles, and much less in the pulp of pear fruits. The intron in the uidA gene stably increased expression in leaves and fruits by approximately twofold. Retransformants with the bar gene showed long-term herbicide resistance and exhibited no consistent changes in leaf size and shape. The transgenic pear was used as rootstock and scion, but grafted plants showed no transport of the GUS protein through the graft in the greenhouse and field. This longest field trial of transgenic fruit trees demonstrates stable expression under varying environmental conditions, the expression-enhancing effect of intron and the absence of unintended effects in single- and double-transformed woody plants.

Pollen-mediated flow of bar gene in transgenic herbicide-resistant turf grass Zoysia japonica

Weed control can be most effectively achieved through the use of herbicide-resistance transgene. A preliminary study bar-transgenic Zoysia japonica posed no serious risk on the unintended escape of the transgene from its cultivation site. The present follow-up investigation of the dispersion of pollen and its short-distance escape outside of the bar-transgenic Zoysia japonica habitats were ascertained in terms of environmental factors affecting anthesis and pollen viability. In a 24-h day cycle, zoysiagrass pollen was released predominantly between 08:00 and 10:00, and the pollen was most viable during the same time interval. Optimal temperature and humidity for pollen viability was 15–20 °C and 80–90%, respectively. The pollen germinated in 120 min after anthesis, but under cloudy conditions germination time doubled. No differences in pollen viability/longevity between the transgenic and non-transgenic plants were observed. The pollen-mediated gene flow of transgenic Zoysia japonica to wild-type non-transgenic zoysiagrass species was monitored by measuring the cross-over rate of the bar gene in the context of three different models. At distances within 5 m, the rate of gene flow ranged from 3 to 5.7% according to the models used. The greater the distance from the transgenic plant site, the lower the gene flow rate. The furthest transgene detected was 38 m away and exhibited a 0.25% gene flow rate. The radial model yielded a 3.7% escape rate within a 3 m radius and was wind direction dependent. The distance- and direction-dependent gene flow events were influenced by wind direction and velocity during flowering season.

How well will stacked transgenic pest/herbicide resistances delay pests from evolving resistance?

Resistance has evolved to single transgenic traits engineered into crops for arthropod and herbicide resistances, and can be expected to evolve to the more recently introduced pathogen resistances. Combining transgenes against the same target pest is being promoted as the solution to the problem. This solution will work if used pre-emptively, but where resistance has evolved to one member of a stack, resistance should easily evolve for the second gene in most cases. We propose and elaborate criteria that could be used to evaluate the value of stacked traits for pest resistance management. Stacked partners must: target the same pest species; be in a tandem construct to preclude segregation; be synchronously expressed in the same tissues; have similar tissue persistence; target pest species that are still susceptible to at least two stacked partners. Additionally, transgene products must not be degraded in the same manner, and there should be a lack of cross-resistance to stacked transgenes or to their products. With stacked herbicide resistance transgenes, both herbicides must be used and have the same persistence. If these criteria are followed, and integrated with other pest management practices, resistance may be considerably delayed. © 2016 Society of Chemical Industry.

A trait stacking system via intra-genomic homologous recombination

A gene targeting method has been developed, which allows the conversion of 'breeding stacks', containing unlinked transgenes into a 'molecular stack' and thereby circumventing the breeding challenges associated with transgene segregation. A gene targeting method has been developed for converting two unlinked trait loci into a single locus transgene stack. The method utilizes intra-genomic homologous recombination (IGHR) between stably integrated target and donor loci which share sequence homology and nuclease cleavage sites whereby the donor contains a promoterless herbicide resistance transgene. Upon crossing with a zinc finger nuclease (ZFN)-expressing plant, double-strand breaks (DSB) are created in both the stably integrated target and donor loci. DSBs flanking the donor locus result in intra-genomic mobilization of a promoterless selectable marker-containing donor sequence, which can be utilized as a template for homology-directed repair of a concomitant DSB at the target locus resulting in a functional selectable marker via nuclease-mediated cassette exchange (NMCE). The method was successfully demonstrated in maize using a glyphosate tolerance gene as a donor whereby up to 3.3% of the resulting progeny embryos cultured on selection medium regenerated plants with the donor sequence integrated into the target locus. The process could be extended to multiple cycles of trait stacking by virtue of a unique intron sequence homology for NMCE between the target and the donor loci. This is the first report that describes NMCE via IGHR, thereby enabling trait stacking using conventional crossing.

Sub-lethal glyphosate exposure alters flowering phenology and causes transient male-sterility in Brassica spp

BackgroundHerbicide resistance in weedy plant populations can develop through different mechanisms such as gene flow of herbicide resistance transgenes from crop species into compatible weedy species or by natural evolution of herbicide resistance or tolerance following selection pressure. Results from our previous studies suggest that sub-lethal levels of the herbicide glyphosate can alter the pattern of gene flow between glyphosate resistant Canola®, Brassica napus, and glyphosate sensitive varieties of B. napus and B. rapa. The objectives of this study were to examine the phenological and developmental changes that occur in Brassica crop and weed species following sub-lethal doses of the herbicides glyphosate and glufosinate. We examined several vegetative and reproductive traits of potted plants under greenhouse conditions, treated with sub-lethal herbicide sprays.ResultsOur results indicate that exposure of Brassica spp. to a sub-lethal dose of glyphosate results in altering flowering phenology and reproductive function. Flowering of all sensitive species was significantly delayed and reproductive function, specifically male fertility, was suppressed. Higher dosage levels typically contributed to an increase in the magnitude of phenotypic changes.ConclusionsThese results demonstrate that Brassica spp. plants that are exposed to sub-lethal doses of glyphosate could be subject to very different pollination patterns and an altered pattern of gene flow that would result from changes in the overlap of flowering phenology between species. Implications include the potential for increased glyphosate resistance evolution and spread in weedy communities exposed to sub-lethal glyphosate.

Morphology and fitness components of wild × crop F1hybrids ofSorghum bicolor(L.) in Ethiopia: implications for survival and introgression of crop genes in the wild pool

Natural hybridization between wild/weedy and crop species often results in rare hybrids, which can be more weedy and difficult to control. Moreover, the advent of transgenic crop plants raises questions of biosafety risk assessment on the consequences of rare hybrids with possible fitness enhancing genes on the environment. This study aimed at measuring the fitness components of wild–crop sorghum hybrids for various juvenile survival and adult morphological and fertility characters as part of the risk assessment of transgenic sorghum in Africa where the crop was believed to have first domesticated and serves as the major staple. Out of a pool of hybrids made in 2010 from 23 wild sorghum accessions and two released cultivated sorghum varieties using hand emasculation techniques, seven were selected for the field study of their fitness components in 2011. The study confirmed that crop–wild hybrids of sorghum are fertile. Two approaches were followed (relative fitness and mid-parent heterosis) which showed that most of the hybrids were as fit as their wild parents, and in some cases they showed mid-parent heterosis for the measured traits. The results of this study highlighted a potential risk that hybrids carrying crop genes (including herbicide resistance transgenes) could pose because they could be more weedy than their wild/weedy parents if transgenic sorghum is deployed in regions where the wild and cultivated sorghum populations coexist, such as in Ethiopia and in other parts of Africa.

A strategy to provide long‐term control of weedy rice while mitigating herbicide resistance transgene flow, and its potential use for other crops with related weeds

Transgenic herbicide-resistant rice is needed to control weeds that have evolved herbicide resistance, as well as for the weedy (feral, red) rice problem, which has been exacerbated by shifting to direct seeding throughout the world-firstly in Europe and the Americas, and now in Asia, as well as in parts of Africa. Transplanting had been the major method of weedy rice control. Experience with imidazolinone-resistant rice shows that gene flow to weedy rice is rapid, negating the utility of the technology. Transgenic technologies are available that can contain herbicide resistance within the crop (cleistogamy, male sterility, targeting to chloroplast genome, etc.), but such technologies are leaky. Mitigation technologies tandemly couple (genetically link) the gene of choice (herbicide resistance) with mitigation genes that are neutral or good for the crop, but render hybrids with weedy rice and their offspring unfit to compete. Mitigation genes confer traits such as non-shattering, dwarfism, no secondary dormancy and herbicide sensitivity. It is proposed to use glyphosate and glufosinate resistances separately as genes of choice, and glufosinate, glyphosate and bentazone susceptibilities as mitigating genes, with a six-season rotation where each stage kills transgenic crop volunteers and transgenic crop x weed hybrids from the previous season.

Evolution of Glyphosate-Resistant Crop Technology

New and improved glyphosate-resistant (GR) crops continue to be rapidly developed. These crops confer greater crop safety to multiple glyphosate applications, higher rates, and wider application timings. Many of these crops will also have glyphosate resistance stacked with traits that confer resistance to herbicides with other modes of actions to expand the utility of existing herbicides and to increase the number of mixture options that can delay the evolution of GR weeds. Some breeding stacks of herbicide resistance traits are currently available, but the trend in the future will be to combine resistance genes in molecular stacks. The first example of such a molecular stack has a new metabolically based mechanism to inactivate glyphosate combined with an active site-based resistance for herbicides that inhibit acetolactate synthase (ALS). This stack confers resistance to glyphosate and all five classes of ALS-inhibiting herbicides. Other molecular stacks will include glyphosate resistance with resistance to auxin herbicides and herbicides that inhibit acetyl coenzyme A carboxylase (ACCase) and 4-hydroxyphenyl pyruvate dioxygenase (HPPD). Scientists are also studying a number of other herbicide resistance transgenes. Some of these new transgenes will be used to make new multiple herbicide-resistant crops that offer growers more herbicide options to meet their changing weed management needs and to help sustain the efficacy of glyphosate.

Do escaped transgenes persist in nature? The case of an herbicide resistance transgene in a weedy Brassica rapa population

The existence of transgenic hybrids resulting from transgene escape from genetically modified (GM) crops to wild or weedy relatives is well documented but the fate of the transgene over time in recipient wild species populations is still relatively unknown. This is the first report of the persistence and apparent introgression, i.e. stable incorporation of genes from one differentiated gene pool into another, of an herbicide resistance transgene from Brassica napus into the gene pool of its weedy relative, Brassica rapa, monitored under natural commercial field conditions. Hybridization between glyphosate-resistant [herbicide resistance (HR)]B. napus and B. rapa was first observed at two Québec sites, Ste Agathe and St Henri, in 2001. B. rapa populations at these two locations were monitored in 2002, 2003 and 2005 for the presence of hybrids and transgene persistence. Hybrid numbers decreased over the 3-year period, from 85 out of approximately 200 plants surveyed in 2002 to only five out of 200 plants in 2005 (St Henri site). Most hybrids had the HR trait, reduced male fertility, intermediate genome structure, and presence of both species-specific amplified fragment length polymorphism markers. Both F(1) and backcross hybrid generations were detected. One introgressed individual, i.e. with the HR trait and diploid ploidy level of B. rapa, was observed in 2005. The latter had reduced pollen viability but produced approximately 480 seeds. Forty-eight of the 50 progeny grown from this plant were diploid with high pollen viability and 22 had the transgene (1:1 segregation). These observations confirm the persistence of the HR trait over time. Persistence occurred over a 6-year period, in the absence of herbicide selection pressure (with the exception of possible exposure to glyphosate in 2002), and in spite of the fitness cost associated with hybridization.

A wheat genomic DNA fragment reduces pollen transmission of maize transgenes by reducing pollen viability

A genomic DNA fragment from wheat carrying the Glu-1Dx5 gene has been shown to exhibit reduced pollen transmission in transgenic maize. To localize the region of the DNA fragment responsible for this reduced pollen transmission, we produced transgenic maize plants in which the wheat genomic DNA proximal to the 1Dx5 coding sequence was replaced with the maize 27 kDa gamma-zein promoter. Like the wheat promoter-driven Glu-1Dx5 transgene, this zein promoter-driven transgene functioned to produce 1Dx5 in maize endosperm. However, with the zein promoter-driven transgene, pollen transmission of the transgene loci was normal in most self- and cross-pollinations. We concluded that the wheat genomic DNA proximal to the wheat 1Dx5 coding sequence was required for reduced pollen transmission of the transgene in maize. In two of four transformation events of the wheat promoter-driven construct examined, pollen exhibited two morphological classes. In one class, pollen was normal in morphology and displayed average viability, and in the second, pollen was reduced in size and did not germinate on artificial media. DNA from the transgene was detectable in mature pollen from plants with reduced pollen transmission of transgene loci. To explain these observations, we hypothesize that elements within the transgene construct interfere with pollen development. We demonstrated that the wheat genomic DNA fragment can be used to control pollen transmission of an herbicide resistance transgene genetically linked to it. The wheat genomic DNA fragment may contain elements that are useful for controlling pollen transmission of transgene loci in commercial maize grain and seed production.

Environmental impacts of transgenic herbicide-resistant crops.

Abstract Transgenic glufosinate- and glyphosate-resistant crops are currently commercialized, and bromoxynil-resistant crops have been removed from the market for economic reasons. Glyphosate-resistant cotton and soyabean have become dominant in those countries where they have been approved for planting. Potential effects of glufosinate and glyphosate on contamination of soil, water and air are minimal, compared to those caused by the herbicides that they replace when herbicide-resistant crops (HRCs) are adopted. No risks have been found with food or feed safety or nutritional value in products from currently available HRCs. Both glufosinate- and glyphosate-resistant crops promote the adoption of reduced- or no-tillage agriculture, reducing environmental degradation by agriculture. HRCs have caused evolution of herbicide resistance in weed species and shifts to those species that are naturally resistant. Transgene flow with HRCs is a concern for commercial and ecological reasons. In canola, rice and bentgrass, herbicide resistance transgenes have been found in fields that are supposed to be non-transgenic. Under some circumstances, transgene flow (introgression) to plants that might become problems in natural ecosystems remains the largest risk of HRCs.

Enhancement of maize transformation efficiency by the use of maize matrix attachment regions

Improving genetic transformation efficiency is a major concern in plant genetic engineering. While various strategies have been investigated, the enhancement of selectable marker gene expression has not been tried extensively. We used maize matrix attachment regions (MARs) to bracket an herbicide resistance transgene, bar. MARs have been reported to enhance transgene expression level and stability. We show here that MARs not only enhance transformation efficiency by 50%, but are also able to increase or decrease relative efficiencies of each step of the regeneration process depending on MAR sequence combinations. Furthermore, we assessed the trans-effect of MARs in co-bombardment experiments with two independent plasmids, one including the MAR sequences and the other one the bar gene. As for simple bombardment, MARs enhanced transformation efficiency by having a positive influence on organogenesis step in the regeneration process.

Lack of stable inheritance of introgressed transgene from oilseed rape in wild radish

Hybridization of Brassica napus L. (oilseed rape) and Raphanus raphanistrum L. (wild radish) has been demonstrated, and may be the first step towards introgression of transgenes in this wild relative. If wild radish were to display a new adaptive advantage by expressing the transgene, this could modify the ecological balance of species within the agro-ecosystem. To determine if transgenes remained stable in the hybrid, the frequency of herbicide resistance was studied over four advanced generations of hybrid progeny (G8 to G11) that were subjected to herbicide selection pressure. It is expected that hemizygous resistant plants containing an herbicide resistance transgene back-crossed to wild radish would have 50% resistant progeny. In each of the G8 to G11 generations, only 18% of the progeny from resistant plants were resistant. The chromosome complement of herbicide-susceptible progenies, analyzed at G9, was not different from that of wild populations of wild radish. Herbicide-resistant G9 progeny showed higher chromosome instability, and one third of the progeny contained a supernumerary chromosome. These results suggest that in the presence of herbicide selection pressure, the transgene for herbicide resistance would be maintained despite a lack of stabilized introgression. In the absence of selection, the frequency of resistance in the population is expected to decline.

Inheritance and segregation of virus and herbicide resistance transgenes in sugarcane.

Transgenic sugarcane parents containing multiple copies of herbicide resistance ( bar) and Sorghum mosaic virus (SrMV) resistance ( hut) genes were crossed with non-transgenic sugarcane varieties. Segregation of the transgenes in the progeny was determined using Southern blot analysis; herbicide resistance and SrMV resistance were assessed using bioassays. The segregation data were used to infer linkage relationships between transgenes in the parent plants. In two of the parents, all transgene insertions were linked in one position in the genome, although some recombination between insertion events did occur. In the third parent, insertion had occurred in two independent, unlinked loci. Analysis of progeny of this parent indicated that rearrangement or mutation occurred in both loci, resulting in non-parental transgene DNA fragments in some progeny. Most transgenic progeny containing the bar gene showed resistance to herbicide. SrMV inoculation indicated that a fairly high proportion of the transgenic progeny showed susceptibility. As the post-transcriptional gene silencing mechanism responsible for the virus resistance phenotype may be reset during meiosis, phenotypic screening of older plants may be a more reliable indication of virus resistance than screening young seedlings. This is the first report of transgene segregation in sugarcane, and we have demonstrated that transgenic sugarcane parents showing stable inheritance of transgenes can be effectively used in breeding programs.

S-1 Herbicide-Resistant Crops-Their Impact on Weed Science

Crops made resistant to herbicides by biotechnology are being widely adopted in North America and other parts of the world. The major impact will be from crops containing transgenes imparting resistance to post-emergence, non-seletctive herbicides for which there is only one commercially successful analogue, such as glyphosate and glufosinate. These products allow the farmer to more effectively use reduced- or no-tillage cultural practices, eliminate use of some of the more environmentally suspect herbicides, and use fewer herbicides to manage nearly the entire spectrum of weed species. In some cases, non-selective herbicides used with herbicide-resistant crops even reduce plant pathogen problems because of their toxicity to some microbes. There is concern among weed scientists that eventual over reliance on fewer weed management strategies will result in evolution of resistance to the more useful herbicides and/or weed population shifts to naturally resistant species. Environmentalists are concerned with gene flow from transgenic crops to closely related wild relatives; however, herbicide resistance transgenes alone confer no fitness advantage outside of fields treated with the herbicide, making it unlikely that they would affect plant populations in natural areas. The next ten years should clarify the eventual impact of these powerful new tools on weed science and weed management.

Herbicide-Resistant Crops-Their Impact on Weed Science.

Crops made resistant to herbicides by biotechnology are being widely adopted in North America and other parts of the world. The major impact will be from crops containing transgenes imparting resistance to post-emergence, non-seletctive herbicides for which there is only one commercially successful analogue, such as glyphosate and glufosinate. These products allow the farmer to more effectively use reduced- or no-tillage cultural practices, eliminate use of some of the more environmentally suspect herbicides, and use fewer herbicides to manage nearly the entire spectrum of weed species. In some cases, non-selective herbicides used with herbicide-resistant crops even reduce plant pathogen problems because of their toxicity to some microbes. There is concern among weed scientists that eventual over reliance on fewer weed management strategies will result in evolution of resistance to the more useful herbicides and/or weed population shifts to naturally resistant species. Environmentalists are concerned with gene flow from transgenic crops to closely related wild relatives; however, herbicide resistance transgenes alone confer no fitness advantage outside of fields treated with the herbicide, making it unlikely that they would affect plant populations in natural areas. The next ten years should clarify the eventual impact of these powerful new tools on weed science and weed management.