Agriculture: the selector of improbable mutations.
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Agriculture↗
Biomedical subjects
Jonathan Gressel
Publications and source records attributed to Jonathan Gressel.
Mitigation of establishment of Brassica napus transgenes in volunteers using a tandem construct containing a selectively unfit gene.
Transgenic oilseed rape (Brassica napus) plants may remain as 'volunteer' weeds in following crops, complicating cultivation and contaminating crop yield. Volunteers can become feral as well as act as a genetic bridge for the transfer of transgenes to weedy relatives. Transgenic mitigation using genes that are positive or neutral to the crop, but deleterious to weeds, should prevent volunteer establishment, as previously intimated using a tobacco (Nicotiana tabacum) model. A transgenically mitigated (TM), dwarf, herbicide-resistant construct using a gibberellic acid-insensitive (Deltagai) gene in the B. napus crop was effective in offsetting the risks of transgene establishment in volunteer populations of B. napus. This may be useful in the absence of herbicide, e.g. when wheat is rotated with oilseed rape. The TM dwarf B. napus plants grown alone had a much higher yield than the non-transgenics, but were exceedingly unfit in competition with non-transgenic tall cohorts. The reproductive fitness of TM B. napus was 0% at 2.5-cm and 4% at 5-cm spacing between glasshouse-grown plants relative to non-transgenic B. napus. Under screen-house conditions, the reproductive fitness of TM B. napus relative to non-transgenic B. napus was less than 12%, and the harvest index of the TM plants was less than 40% of that of the non-transgenic competitors. The data clearly indicate that the Deltagai gene greatly enhances the yield in a weed-free transgenic crop, but the dwarf plants can be eliminated when competing with non-transgenic cohorts (and presumably other species) when the selective herbicide is not used.
Brassica napus↗
Mitigation using a tandem construct containing a selectively unfit gene precludes establishment of Brassica napus transgenes in hybrids and backcrosses with weedy Brassica rapa.
Transgenic oilseed rape (Brassica napus) plants can interbreed with nearby weedy Brassica rapa, potentially enhancing the weediness and/or invasiveness of subsequent hybrid offspring. We have previously demonstrated that transgenic mitigation effectively reduces the fitness of the transgenic dwarf and herbicide-resistant B. napus volunteers. We now report the efficacy of such a tandem construct, including a primary herbicide-resistant gene and a dwarfing mitigator gene, to preclude the risks of gene establishment in the related weed B. rapa and its backcross progeny. The transgenically mitigated and non-transgenic B. rapa x B. napus interspecific hybrids and the backcrosses (BC(1)) with B. rapa were grown alone and in competition with B. rapa weed. The reproductive fitness of hybrid offspring progressively decreased with increased B. rapa genes in the offspring, illustrating the efficacy of the concept. The fitness of F(2) interspecific non-transgenic hybrids was between 50% and 80% of the competing weedy B. rapa, whereas the fitness of the comparable T(2) interspecific transgenic hybrids was never more than 2%. The reproductive fitness of the transgenic T(2) BC(1) mixed with B. rapa was further severely suppressed to 0.9% of that of the competing weed due to dwarfism. Clearly, the mitigation technology works efficiently in a rapeseed crop-weed system under biocontainment-controlled environments, but field studies should further validate its utility for minimizing the risks of gene flow.
Brassica napus↗
Metabolic engineering of plant cells for biotransformation of hesperedin into neohesperidin, a substrate for production of the low-calorie sweetener and flavor enhancer NHDC.
Neohesperidin dihydrochalcone (NHDC) is a seminatural, safe, low-calorie sweetener, bitterness blocker, and flavor enhancer with unique properties and applications for the food, beverage, pharmaceutical, and animal feed industries. Current production is limited by the availability of the substrate neohesperidin, a flavonoid that accumulates to significant levels only in the inedible bitter citrus species. We propose a process to convert hesperidin, a tasteless flavonoid extracted from orange peels that are abundant byproducts of the vast orange juice industry, into neohesperidin using metabolic engineering and biotransformation via three steps: (i) extraction of hesperidin from orange peels, (ii) hydrolysis of sugar moieties, and (iii) biotransformation of hesperidin hydrolysis products into neohesperidin. We overcame the current technological bottleneck in biotransformation of hesperidin hydrolysis products into neohesperidin using metabolically engineered plant cell cultures expressing a recombinant flavanone-7-O-glucoside-2-O-rhamnosyltransferase. A small-scale production experiment established the feasibility of the proposed process.
Cells, Cultured↗
Sequence evidence for sporadic intergeneric DNA introgression from wheat into a wild Aegilops species.
Introgressive hybridization has played a crucial role in the evolution of many plant species, especially polyploids. The duplicated genetic material and wide geographical distribution facilitate hybridization and introgression among polyploid species having either homologous or homoeologous genomes. Such introgression may lead to the production of recombinant genomes that are more difficult to form at the diploid level. Crop genes that have introgressed into wild relatives can increase the capability of the wild relatives to adapt to agricultural environments and compete with crops or to compete with other wild species. Although the transfer of genes from crops into their conspecific immediate wild progenitors has been reported, little is known about spontaneous gene movement from crops to more distantly related species. We describe recent spontaneous DNA introgression from domesticated polyploid wheat into distantly related, wild tetraploid Aegilops peregrina (syn. Aegilops variabilis) and the stabilization of this sequence in wild populations despite not having homologous chromosomes. Our results show that DNA can spontaneously introgress between homoeologous genomes of species of the tribe Triticeae and, in the case of crop-wild relatives, possibly enrich the wild population. These results also emphasize the need for fail-safe mechanisms in transgenic crops to prevent gene flow where there may be ecological risks.
Base Sequence↗
Poor competitive fitness of transgenically mitigated tobacco in competition with the wild type in a replacement series.
Transgenic crops can interbreed with other crop cultivars or with related weeds, increasing the potential of the hybrid progeny for competition. To prevent generating competitive hybrids, we previously tested tobacco (Nicotiana tabacum L.) as a model for validating the transgenic mitigation (TM) concept using tandem constructs where a gene of choice is linked to mitigating genes that are positive or neutral to the crop, but deleterious to a recipient under competition. Here, we examine the efficacy of the TM concept at various ratios of transgenically mitigated tobacco in competition with the wild type tobacco in an ecological replacement series. The dwarf/herbicide-resistant TM transgenic plants cultivated alone under self-competition grew well and formed many more flowers than the tall wild type, which is an indication of greater reproductivity. In contrast to the wild type, TM flowering was almost completely suppressed in mixed cultures at most TM/wild type ratios up to 75% transgenic, as the TM plants were extremely unfit to reproduce. In addition, homozygous TM progeny had an even lower competitive fitness against the wild type than hemizygous/homozygous TM segregants. Thus, the TM technology was effective in reducing the risk of transgene establishment of intraspecific transgenic hybrids at different competitive levels, at the close spacing typical of weed populations.
Arabidopsis Proteins↗
Tandem constructs to mitigate transgene persistence: tobacco as a model.
Some transgenic crops can introgress genes into other varieties of the crop, to related weeds or themselves remain as 'volunteer' weeds, potentially enhancing the invasiveness or weediness of the resulting offspring. The presently suggested mechanisms for transgene containment allow low frequency of gene release (leakage), requiring the mitigation of continued spread. Transgenic mitigation (TM), where a desired primary gene is tandemly coupled with mitigating genes that are positive or neutral to the crop but deleterious to hybrids and their progeny, was tested as a mechanism to mitigate transgene introgression. Dwarfism, which typically increases crop yield while decreasing the ability to compete, was used as a mitigator. A construct of a dominant ahasR (acetohydroxy acid synthase) gene conferring herbicide resistance in tandem with the semidominant mitigator dwarfing Delta gai (gibberellic acid-insensitive) gene was transformed into tobacco (Nicotiana tabacum). The integration and the phenotypic stability of the tandemly linked ahasR and Delta gai genomic inserts in later generations were confirmed by polymerase chain reaction. The hemizygous semidwarf imazapyr-resistant TM T1 (= BC1) transgenic plants were weak competitors when cocultivated with wild type segregants under greenhouse conditions and without using the herbicide. The competition was most intense at close spacings typical of weed offspring. Most dwarf plants interspersed with wild type died at 1-cm, > 70% at 2.5-cm and 45% at 5-cm spacing, and the dwarf survivors formed no flowers. At 10-cm spacing, where few TM plants died, only those TM plants growing at the periphery of the large cultivation containers formed flowers, after the wild type plants terminated growth. The highest reproductive TM fitness relative to the wild type was 17%. The results demonstrate the suppression of crop-weed hybrids when competing with wild type weeds, or such crops as volunteer weeds, in seasons when the selector (herbicide) is not used. The linked unfitness would be continuously manifested in future generations, keeping the transgene at a low frequency.
Acetolactate Synthase↗
Citrus fruit bitter flavors: isolation and functional characterization of the gene Cm1,2RhaT encoding a 1,2 rhamnosyltransferase, a key enzyme in the biosynthesis of the bitter flavonoids of citrus.
Species of the genus Citrus accumulate large quantities of flavanones that affect fruit flavor and have been documented to benefit human health. Bitter species, such as grapefruit and pummelo, accumulate bitter flavanone-7-O-neohesperidosides responsible, in part, for their characteristic taste. Non-bitter species, such as mandarin and orange, accumulate only tasteless flavanone-7-O-rutinosides. The key flavor-determining step of citrus flavanone-glycoside biosynthesis is catalyzed by rhamnosyltransferases; 1,2 rhamnosyltransferases (1,2RhaT) catalyze biosynthesis of the bitter neohesperidosides, while 1,6 rhamnosyltransferases (1,6RhaT) catalyze biosynthesis of the tasteless rutinosides. We report on the isolation and functional characterization of the gene Cm1,2RhaT from pummelo which encodes a citrus 1,2RhaT. Functional analysis of Cm1,2RhaT recombinant enzyme was conducted by biotransformation of the substrates using transgenic plant cell culture. Flavanones and flavones, but not flavonols, were biotransformed into 7-O-neohesperidosides by the transgenic BY2 tobacco cells expressing recombinant Cm1,2RhaT. Immunoblot analysis established that 1,2RhaT protein was expressed only in the bitter citrus species and that 1,6RhaT enzyme, whose activity was previously documented in non-bitter species, was not cross-reactive. Expression of Cm1,2RhaT at the RNA level was prominent in young fruit and leaves, but low in the corresponding mature tissue, thus correlating well with the developmental pattern of accumulation of flavanone-neohesperidosides previously established. Phylogenetic analysis of the flavonoid glycosyltransferase gene family places Cm1,2RhaT on a separate gene cluster together with the only other functionally characterized flavonoid-glucoside rhamnosyltransferase gene, suggesting a common evolutionary origin for rhamnosyltransferases specializing in glycosylation of the sugar moieties of flavonoid glucosides.
Amino Acid Sequence↗
Ultralow calcium requirements of fungi facilitate use of calcium regulating agents to suppress host calcium-dependent defenses, synergizing infection by a mycoherbicide.
Infection by many fungi activates a variety of calcium dependent defenses in the hosts, slowing or suppressing the attacker and limiting the efficacy of mycoherbicides. The calcium requirement for fungal growth is so low that it could only be implied based on fungi containing calcium-dependent signaling enzymes. Analytical grade media contain <2 microM calcium, and the addition of specific chelators does not affect fungal growth. Hydrophobic derivatives of the calcium-specific chelator BAPTA designed to traverse plant cuticles were synthesized in order to chelate calcium internally during fungal attack. Some chelators as well as calcium precipitating oxalate and channel blocker verapamil were applied with a weakly mycoherbicidal Colletotrichum coccodes to cotyledons of compatible Abutilon threophrasti. They suppressed calcium dependent callose biosynthesis in the weed and increased virulence but may have affected other calcium-dependent processes that facilitate virulence. The low calcium requirement of fungi, and their high affinity for calcium, allows the application of calcium-regulating agents as synergists for mycoherbicides where the weed uses calcium-dependent defenses.
Calcium↗
Engineering hypervirulence in a mycoherbicidal fungus for efficient weed control.
Agents proposed for biocontrol of major weeds in arable row-crop agriculture have not met expectations because an evolutionary balance has developed between microorganism and weed, even when the mycoherbicide is used inundatively at very high levels (>10(4)spores/cm<(2)). Sufficient virulence can be achieved by transferring genes to the microorganism, tipping the evolutionary balance. Virulence was increased ninefold and was more rapidly effected; furthermore, the requirement for a long duration at high humidity was decreased by introducing NEP1 encoding a phytotoxic protein, to an Abutilon theophrasti-specific, weakly mycoherbicidal strain of Colletotrichum coccodes. The parent strain was at best infective on juvenile cotyledons of this intransigent weed. The transgenic strain was lethal through the three-leaf stage, a sufficient time window to control this asynchronously germinating weed. Strategies of coupling virulence genes with fail-safe mechanisms to prevent spread (due to broadened host range) and to mitigate transgene introgression into crop pathogens could be very useful in the biocontrol of major weeds in row crops.
Agriculture↗
Transformation of carrots with mutant acetolactate synthase for Orobanche (broomrape) control.
Parasitic Orobanche spp are major constraints to vegetable crop production in the Mediterranean basin (to eastern Europe) and in localized places in India, China and the USA. Transgenic target-site herbicide resistance (eg, to acetolactate synthase inhibitors) allows for movement of unmetabolized herbicide through the crop to the photosynthate sink in the parasite, as well as through the soil. We report the successful engineering of a mutant acetolactate synthase (ALS) gene into carrot, allowing control of broomrape already in heterozygotes of the first back-crossed generation, by imazapyr, an imidazolinone ALS inhibitor. It is expected that homozygotes will have higher levels of resistance.
Acetolactate Synthase↗
Universal inheritable barcodes for identifying organisms.
The needs for recognition of novel conventional or transgenic organisms include protection of patented or Identity Preserved lines, detecting transgenics and tracing dispersal. We propose simple 'Biobarcodes' using universal PCR primers to recognize the universal 'nonsense' recognition site of all biobarcodes, followed by a variable nonsense sequence. The proposed sequences are long enough to allow recognition in spite of mutations, have stop codons to prevent coding, and will not self anneal. Sequences of PCR-amplified biobarcodes can be compared to a universal database.
DNA, Plant↗
Infection of tubercles of the parasitic weed Orobanche aegyptiaca by mycoherbicidal Fusarium species.
Progression of the infection by host-specific strains of Fusarium oxysporum and Fusarium arthrosporioides of Orobanche aegyptiaca (Egyptian broomrape) tubercles attached to tomato roots was tracked using light, confocal and electron microscopy. Mycelia transformed with the gene for green fluorescent protein were viewed using a confocal microscope. Fungal penetration was preceded by a rapid loss of starch, with approx. 10 % remaining at 9 h and no measurable starch at 24 h. Penetration into the Orobanche tubercles began by 12 h after inoculation. Hyphae penetrated the outer six cell layers by 24 h, reaching the centre of the tubercles by 48 h and infecting nearly all cells by 72 h. Most of the infected tubercles were dead by 96 h. Breakdown of cell walls and the disintegration of cytoplasm in and around the infected cells occurred between 48 and 96 h. Lignin-like material increased in tubercle cells of infected tissues over time, but did not appear to be effective in limiting fungal penetration or spread. Callose, suberin, constitutive toxins and phytoalexins were not detected in infected tubercles, suggesting that there are no obvious defence mechanisms to overcome. Both Fusarium spp. pathogenic on Orobanche produced fumonisin-like ceramide synthase inhibitors, while fusaric acid was produced only by F. oxysporum in liquid culture. The organisms do not have sufficient virulence for field use (based on glasshouse testing), suggesting that virulence should be transgenically enhanced or additional isolates sought.
Fusarium↗
Assessing and managing biological risks of plants used for bioremediation, including risks of transgene flow.
The plants used for phytoremediation pose special biological risks, whether transgenic or not, as most of the species: (a) are semi-domesticated; (b) are introduced from other habitats; (c) can become established in the contaminated site; (d) can spread and displace native species, and/or; (e) may introgress transgenes into related species. The addition of transgenes can reduce the risks, e.g. to sterilize or render the species and hybrid offspring hypersensitive to environmental effects (heat, cold), or to a chemical that will cull the species. Various measures can contain transgenes used in phytoremediation species to prevent gene flow, but most containment technologies are both uni-directional (prevent either outflow or influx), and are inherently leaky, even a concept specifically utilizable for phytoremediation--grafting non-transgenic scions on bioremediating transgenic rootstocks. Containment mechanisms should be either stacked with each other or with "mitigator" genes. Transgenic mitigation (TM) has mitigator genes added in tandem to the desired primary transgene (genetically linked) and the mitigator genes confer traits that are positive or neutral to the desired species but are deleterious to hybrids, keeping them at very low frequencies. The concept was demonstrated in tobacco and oilseed rape with a dwarfing mitigator gene that enhanced the reproductive productivity (harvest index) when cultured alone, but eliminated it from mixed populations. Besides the mitigator genes previously proposed for crop species (sterility, no seed shattering, dwarfing, no secondary dormancy) there are genes especially appropriate for phytoremediation, e.g. overexpression of cytokinin oxidase (reduces cytokinin levels) conferring reduced shoot systems (unfitness to compete) with a more extensive root system that is better for extracting toxic wastes as well as no-flowering for vegetatively propagated species. Thus, biotechnology can be harnessed to reduce risks from both non-transgenic and transgenic phytoremediation species.
Biodegradation, Environmental↗