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Evolved resistance and regulatory deregistration have severely limited farmers' pesticide options. Many potential new pesticide target sites have been elucidated using targeted gene suppression and mutational tools, but few small molecules could be found that inhibit the target enzymes; the targets were considered 'undruggable'. Some organisms from all biological kingdoms use toxic peptides to ward off or kill enemies, and the agrochemical industry has used a few peptide analogs (glufosinate and bialophos) for field application. Conversely, pharmaceutical scientists have been using three-dimensional target protein structure to discover and synthesize short peptides that bind tightly to the surfaces of, and inhibit previously undruggable targets. New computational tools to quickly elucidate 3-D protein structure from amino acid sequence have just emerged. They replace crystallizing target proteins and performing X-ray crystallography to elucidate 3-D structure. These new tools allow prediction of peptides that will bind to the target proteins. They have further modified such peptides to enhance penetration, translocation and temperature stability. There is reason to assume that the same pioneering techniques can be used to develop peptide pesticides as well as pesticide synergists that act against undruggable targets and have excellent environmental and toxicological profiles. © 2022 Society of Chemical Industry.

Microbiome organisms can degrade environmental xenobiotics including pesticides, conferring resistance to most types of pests. Some cases of pesticide resistance in insects, nematodes and weeds are now documented to be due to microbiome detoxification, and is a demonstrated possibility with rodents. Some cases of metabolic resistance may have been misattributed to pest metabolism, and not to organisms in the microbiome, because few researchers use axenic pests in studying pesticide metabolism. Instances of microbiomes evolving pesticide resistance contributing to resistance of their hosts may become more common due the erratic nature of climate change, as microbiome populations typically increase and evolve faster in stressful conditions. Conversely, microbiome organisms can be engineered to provide crops and beneficial insects with needed resistance to herbicides and insecticides, respectively, but there has not been sufficient efficacy to achieve commercial products useful at the field level, even with genetically engineered microbiome organisms. © 2017 Society of Chemical Industry.

Liver-affecting, carcinogenic aflatoxins produced by Aspergillus spp. are a major problem, especially in the humid developing world where storage conditions are often optimal for the fungi. Peanuts and maize have been transformed with RNAi constructs targeting Aspergillus flavus polyketide-synthase, an early key enzyme in aflatoxin biosynthesis. Aflatoxin biosynthesis was suppressed in developing immature grain, less so in late maturing grain, and it is doubtful that the technology will be effective in near dry mature grain. The infected grain was still mouldy. As Aspergillus that infects grain preharvest can continue to grow and produce aflatoxin in poorly stored grain, and grain storage insects vector further infections, this technology seems to have little potential utility in the humid tropics. The biotechnological approaches of RNAi directly targeting Aspergillus, coupled with transgenic insecticidal proteins should be far more effective. These biotechnological approaches can be used in tandem with the RNAi against polyketide-synthase, as well as with irradiation, biocontrol and better grain drying and hermetic dry storage in a controlled atmosphere. © 2017 Society of Chemical Industry.

For many years, the emphasis of industry discovery programs has been on finding new target sites of pesticides and finding pesticides that inhibit single targets. There had been an emphasis on genomics in finding single targets for potential pesticides. There is also the claim that registration of single target inhibiting pesticides is simpler if the mode of action is known. Conversely, if one looks at the evolution of resistance from an epidemiological perspective to ascertain which pesticides have been the most recalcitrant to evolutionary forces, it is those that have multiple target sites of action. Non-target-site resistances can evolve to multi-target-site inhibitors, but these resistances can often be overcome by structural modification of the pesticide. Industry has looked at pest-toxic natural products as pesticide leads, but seems to have abandoned those where they can find no single target of action. Perhaps nature has been intelligent and evolved many natural products that are synergistic multi-target-site inhibitors, and that is why natural compounds have been active for millennia? We should be learning from nature while combining new chemistry technologies with vast accrued databases and computer aided design allowing fragment-based discovery and scaffold hopping to produce multi-target site inhibitors instead of single target pesticides. © 2019 Society of Chemical Industry.

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

We speculate that multicopy transposons, carrying both fitness and unfitness genes, can provide new positive and negative selection options to intractable weed problems. Multicopy transposons rapidly disseminate through populations, appearing in approximately 100% of progeny, unlike nuclear transgenes, which appear in a proportion of segregating populations. Different unfitness transgenes and modes of propagation will be appropriate for different cases: (1) outcrossing Amaranthus spp. (that evolved resistances to major herbicides); (2) Lolium spp., important pasture grasses, yet herbicide-resistant weeds in crops; (3) rice (Oryza sativa), often infested with feral weedy rice, which interbreeds with the crop; and (4) self-compatible sorghum (Sorghum bicolor), which readily crosses with conspecific shattercane and with allotetraploid johnsongrass (Sorghum halepense). The speculated outcome of these scenarios is to generate weed populations that contain the unfitness gene and thus are easily controllable. Unfitness genes can be under chemically or environmentally inducible promoters, activated after gene dissemination, or under constitutive promoters where the gene function is utilized only at special times (e.g. sensitivity to an herbicide). The transposons can be vectored to the weeds by introgression from the crop (in rice, sorghum, and Lolium spp.) or from planted engineered weed (Amaranthus spp.) using a gene conferring the degradation of a no longer widely used herbicide, especially in tandem with an herbicide-resistant gene that kills all nonhybrids, facilitating the rapid dissemination of the multicopy transposons in a weedy population.

Cultivating algae on a large scale will inevitably lead to spills into natural ecosystems. Most risk analyses have dealt only with transgenic algae, without considering the risks of cultivating the corresponding non-transgenic wild type species. This is despite the long-studied 'paradox of the plankton', which describes the unsuitability of laboratory experimentation or modeling to predict the outcome of introducing non-native algae into a new ecosystem. Risk analyses of transgenic strains of native algae can be based on whether they are more fit or less fit than their wild type, but these are not possible with non-native species. Risks from spills can be minimized by mutagenically or transgenically deleting genes that are unnecessary in culture but obligatory in nature.

Sub-toxic doses of many toxicants have positive, beneficial effects on productivity, or stress resistance (hormesis). Transcriptomic, proteomic, and metabolomic responses to a disparate variety hormetic agents, coupled with bioinformatic analyses, can be used to identify consensus genes, their controlling elements, and their metabolites related to stimulation of growth and/or health. This information can then be used as a method for generating healthier and higher yielding crops using transgenic or other biotechnological techniques. The same bioinformatic information can be used to develop knowledge-based, transcriptomic, proteomic and metabolomic high throughput pre-screens using young plants to identify hormetic chemicals that are potentially useful for enhancement of crop health and yield. Such pre-screens preclude the need to use whole plants through maturity. While the hormetic effectors themselves have to date been of limited direct utility, it is clear that they can be used to help pinpoint genes and chemicals that are potentially useful. This is superior to the presently used random screening or even “educated guess” screening of genes and chemicals.

As there are industrial and societal interests in cultivating algae on a large scale, there inevitably will be spills of cultured algae into natural ecosystems. The assessment of environmental risks of such spills is especially hard for cultured, non-native microalgae species due to the “paradox of the plankton”, the paradox that large numbers of species with varying degrees of fitness co-exist in natural ecosystems in an unpredictable, fluctuating species balance. The risk analysis may be more straightforward for special cases, e.g. transgenic or mutated strains of common, indigenous species because their behavior can be compared to their wild types. Risk assessment can be based on presently used Good Industrial Large Scale Practice considerations, particularly the use of mitigating traits that severely decrease fitness. Some desirable introduced genes may have some unfitness in natural ecosystems and can be coupled with antisense or RNAi suppressed genes to mitigate genes that increase fitness. The most stringent mitigation systems are needed especially for non-native species and can utilize deletion mutations (e.g. in carbon capture or nitrate utilization) that allow cultivation only in artificial systems and are lethal to the algae in nature.

There are two major energy and cost constraints to bulk production of single cell microalgae for biofuels or feed: expensive culture systems with high capital costs and high energy requirements for mixing and gas exchange; and the cost of harvesting using high-speed continuous centrifugation for dewatering. This report deals with the latter; harvesting by flocculation where theory states that alkaline flocculants neutralize the repelling surface charge of algal cells, allowing them to coalesce into a floc. It had been assumed that with such electrostatic flocculation, the more cells to be flocculated, the more flocculant needed, in a linear stoichiometric fashion, rendering flocculation overly expensive. Counter to theory of electrostatic flocculation, we find that the amount of alkaline flocculant needed is a function of the logarithm of cell density, with dense cultures requiring an order of magnitude less base than dilute suspensions, with flocculation occurring at a lower pH. Various other theories abound that flocculation can be due to multi-valent cross-linking, or co-precipitation with phosphate or with magnesium and calcium, but are clearly not relevant with the flocculants we used. Monovalent bases that cannot cross-link or precipitate phosphate work with the same log-linear stoichiometry as the divalent bases, obviating those theories, leaving electrostatic flocculation as the only tenable theory of flocculation with the materials used. The cost of flocculation of dense cultures with this procedure should be below $1.00/T algae for mixed calcium:magnesium hydroxides.

The containment strategies to keep transgenes within a crop, and to keep transgenic crop seed (or hybrids between transgenic crops and their closely related nontransgenic neighbors) from spreading are not absolute. Containers eventually leak, and some containment systems are inherently leaky. Once there is a leak, the containment strategies cannot stop the spread. Some containment methods described in other chapters are unilateral, they prevent gene flow in one direction only (and are not even near absolute in that direction). For example, if a transgene is chloroplast encoded, and the pollen of the species does not transmit chloroplast DNA, the relative can cross with the crop, and its hybrid and backcrosses can be the recurrent pollen parent, moving the transgene to the relative. Additionally, even in species where there is maternal inheritance, plastid encoded traits do manage to be pollen transmitted by about 0.5% of pollen grains (Wang et al., 2004; Svab andMaliga 2007). Crops can be engineered to lack pollen production and be male sterile (rarely 100%) (Moon et al., 2010), but the wild or weedy relative can pollinate the crop. If various containment systems such as the genetic use restriction technologies (GURTs) that are discussed in other chapters are absolute, there still can be pollen transmission in the seed production fields before the GURTs are activated. Some seeds from transgenic crops are typically not harvested. If they persist to the next growing seasons, they will become “volunteer weeds” in the rotational crops that follow. There is selection pressure on such volunteer weeds to lose some of the recessive domestication traits such as uniform germination (lack of secondary dormancy that hedges chances against being controlled during the following and subsequent seasons), lack of seed shattering, and in green revolution crops, dwarfism. The feral traits are mostly dominant back mutations from crop to weedy traits such as shattering, dormancy, and height. They render the volunteer cum feral weeds more competitive with their domesticated cohorts, as well as with other weeds. The stress of being a volunteer weed in an agroecosystem may actually facilitate a more rapid evolution of back mutations to ferality, as stresses can increase the rate of mutations (Gressel and Levy, 2009). Thus, there is a need to have a second line of defense after containment to mitigate against genetic leaks from establishing in the field, whether leaks are of pollen or seed.

Strigais a major constraint to food production in Africa. Most technologies developed for the eradication ofStriga asiaticafrom the United States are not adaptable to Africa. Imazapyr and pyrithiobac coated imidazolinone-resistant (IR)-resistant maize seed prior to planting at rates of 30 to 45 g ha−1provide near season long control ofStrigaand can increase maize yields three- to fourfold if supplied with fertilizer. Slow release seed coatings reduce maize injury when post-planting rains are sparse and improveStrigacontrol when there is excessive rainfall early in the season. Models suggest that herbicide resistance may not be a significant threat in short season maize, but vigilance in removing floweringStrigaplants that are not controlled is recommended due to the known risk of evolution of resistance to these herbicides. Stacking the IR gene with glyphosate resistance and using imazapyr treated seed and applying glyphosate mid-season would provide season longStrigacontrol and delay the evolution of resistance to both herbicides. To date, adoption of this technology has been limited by a number of factors. However, it should be included as one component of a multi-factor approach to increasing maize productivity in areas of Africa whereStrigais problematic.

At very low pesticide rates, a certain low proportion of pests may receive a sublethal dose, are highly stressed by the pesticide and yet survive. Stress is a general enhancer of mutation rates. Thus, the survivors are likely to have more than normal mutations, which might include mutations leading to pesticide resistance, both for multifactorial (polygenic, gene amplification, sequential allelic mutations) and for major gene resistance. Management strategies should consider how to eliminate the subpopulation of pests with the high mutation rates, but the best strategy is probably to avoid too low application rates of pesticides from the outset.

There are a few instances where seed-expressed traits may pose a biosafety risk, depending on the species, the trait, and where it is cultivated. The fewest ecological risks of pollen flow are posed to the natural environment, as there are few crops growing in or near pristine environments that have interbreeding relatives, or bear traits that could disrupt ecological balances. Some seed-expressed traits might pose an agro-ecological risk if introgressed into related weeds. Only pharmaceutical or industrial seed-expressed traits are likely to introgress into other varieties of a crop at regulatory red flag levels due to zero tolerance at the most sensitive levels of detection when grown at normal separation distances. Other traits would be below regulatory thresholds if required separations distances are met. Two of the three transgenic containment methods described (plastome transformation, attenuated viruses) are unlikely to work with seed-expressed traits. Transgenic mitigation could be used where the seed-expressed trait is linked with a trait that is positive or neutral to a crop but deleterious to its wild or weedy relative. Typical mitigation traits are dwarfing, no seed shattering, and lack of secondary dormancy. There are special mitigator genes, such as transgenically dominant sugary endosperm that might be appropriate for pharmaceutical traits. Biosafety gene flow protection mechanisms are also needed to prevent wild type poisonous seed crops (e.g. castor and Jatropha) from introgressing toxin genes into seeds of varieties where these toxins have been transgenically eliminated.

SUMMARYWeeds have been controlled so successfully by herbicides, subsequently supplemented by transgenic herbicide-resistant crops, that past intractable problems have been ignored and new ones are evolving. However, industry-led discovery of new herbicide targets is now lacking and weed science as a discipline is contracting globally. This paper describes novel technologies for dealing with major problems, including: better understanding of weed biology coupled with genomics; novel herbicide-resistant crops as well as engineered weed-competing crops; multi-target herbicides; and enhanced biocontrol agents. Together, these approaches may comprise the components of future integrated packages to slow down the evolution of new weed problems.

Except for a small number of cases in which biocontrol agents were introduced from the site of origin of a weed (classical biocontrol), there have been few cases where a pathogen was virulent enough to perform cost effectively in the field as a mycoherbicide. Mycoherbicides are typically weed species specific, so compatibility with herbicides used to control other weeds is often studied. There can be a synergy between mycoherbicides and herbicides at the field level due to overlapping weed spectra (such synergies are not discussed in depth herein). Two approaches have been used to ascertain whether there is synergy in controlling the target weed: (1) random screening with herbicides; (2) using herbicides as antimetabolites to inhibit specific pathways, enhancing virulence. Glyphosate is the most common herbicide to synergize mycoherbicides, possibly due to its dual function as an inhibitor of biosynthesis of phenylpropanoid phytoalexins by suppressing enolphosphate-shikimate phosphate synthase, or by suppressing callose production (by inhibiting callose synthase) as well as inhibiting other calcium-dependent pathways due to the calcium-chelating properties of glyphosate.

A greater number of, and more varied, modes of resistance have evolved in weeds than in other pests because the usage of herbicides is far more extensive than the usage of other pesticides, and because weed seed output is so great. The discovery and development of selective herbicides are more problematic than those of insecticides and fungicides, as these must only differentiate between plant and insect or pathogen. Herbicides are typically selective between plants, meaning that before deployment there are already some crops possessing natural herbicide resistance that weeds could evolve. The concepts of the evolution of resistance and the mechanisms of delaying resistance have evolved as nature has continually evolved new types of resistance. Major gene target-site mutations were the first types to evolve, with initial consideration devoted mainly to them, but slowly 'creeping' resistance, gradually accruing increasing levels of resistance, has become a major force owing to an incremental accumulation of genetic changes in weed populations. Weeds have evolved mechanisms unknown even in antibiotic as well as other drug and pesticide resistances. It is even possible that cases of epigenetic 'remembered' resistances may have appeared.

The greatest cost to the farmer in time and variable costs is control of weeds (Highly Invasive Vegetation = HIV). Despite farmer efforts, weeds still result in the greatest crop losses of all biotic constraints. The costs due to this HIV are greatest in the parts of the developing world where manual labor (more typically “femanual”) is used, and the populations are becoming more aged (due to youth abandoning agriculture), or more feeble due to debilitating diseases such as malaria and HIV-AIDS. Two case studies are presented: the previously intractable problems with Striga (witchweed) species in Africa; and rice cultivation and the emerging problems with the labor-saving shift to direct seeding, which has resulted in outbreaks of a weedy/feral form of rice, as well as herbicide-resistant Echinochloa spp. Biotechnology has much to offer as part of the solution to these major HIV problems, whether as transgenic herbicide resistant crops, or as weed resistant crops or through transgenically enhanced weed-specific biocontrol agents. Where necessary, transgenic tricks will also be needed in many cases to prevent transgene flow from crop to related weedy relatives, and possible technologies are described. Ever since the advent of agriculture, solutions to crop protection problems have been effective but ephemeral, and new solutions will be needed in the future. Using mixed solutions leads to solutions lasting synergistically longer, so efforts should be made to use mixed technologies to extend the lifetime of the next generation of sorely needed solutions to the problems of HIV.

Imidazolinone resistant maize seed dressed with imazapyr has been successfully used to control the parasitic weed Striga in many locations, and has begun to be commercially successful in Africa. Despite this, occasionally poor effectiveness of control had been documented in some sites that required explanation. Analysis of the data against rainfall patterns suggested that: 1. poor maize emergence occurred when there was limited rainfall at germination, possibly due to inhibition by a very high herbicide concentration too near the maize seed; 2. there was poor Striga control in seasons of very high rainfall, possibly due to herbicide washout away from the maize root zone where Striga germinates and attaches. These field assessments were matched by experiments suggesting that slow release formulations might alleviate the problems. A series of slow release formulations were synthesized based on binding imazapyr to high capacity anion exchangers and using them to coat maize seed. The best seems to be a polyethyleneimine gel. Epidemiological field data from a multitude of sites support the conclusion that the slow release formulations increased stand establishment across sites and seasons compared to the control where there was low rainfall.

Abutilon theophrasti (Malvaceae) is a widely distributed weed that infests a variety of agricultural crops. Cotton is in the same botanical family, precluding the use of selective chemical herbicides. An Abutilon -specific pathovar of Colletotrichum coccodes has been considered as a potential bioherbicide for the selective control of A. theophrasti . An evolutionary balance between microorganism and weed maintains a relatively low virulence of the microorganism, such that it does not destroy its food supply by rapidly killing its host. We overexpressed the oah A (oxaloacetate acetylhydrolase) gene in C. coccodes to increase oxalate production, as oxalate inhibits plant-defensive callose synthase by chelating calcium, an obligate co-factor of this enzyme. C. coccodes oahA transformants acidified the surrounding medium, an indication of acid production by hydrolysis of oxaloacetate. Less callose was seen visually around lesions caused by C. coccodes oahA transformants than the wild type in preliminary experiments, but this was not biochemically quantifiable. Further optimization might be obtained through gene stacking, or the addition of multiple virulence genes to this potential biocontrol agent.

It is necessary to control root parasitic weeds before or as they attach to the crop. This can only be easily achieved chemically with herbicides that are systemic, or with herbicides that are active in soil. Long-term control can only be attained if the crops do not metabolise the herbicide, i.e. have target-site resistance. Such target-site resistances have allowed foliar applications of herbicides inhibiting enol-pyruvylshikimate phosphate synthase (EPSPS) (glyphosate), acetolactate synthase (ALS) (e.g. chlorsulfuron, imazapyr) and dihydropteroate synthase (asulam) for Orobanche control in experimental conditions with various crops. Large-scale use of imazapyr as a seed dressing of imidazolinone-resistant maize has been commercialised for Striga control. Crops with two target-site resistances will be more resilient to the evolution of resistance in the parasite, if well managed.

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.

Durum and bread wheat need transgenic traits such as herbicide and disease resistance due to recent evolution of herbicide resistant grass weeds and an intractable new strain of stem rust. Transgenic wheat varieties have not been commercialized partly due to potential transgene movement to wild/weedy relatives, which occurs naturally to closely related Aegilops and other spp. Recombination does not occur in the F 1 hybrid between wheat and its relatives due to the presence of the Ph 1 gene on wheat chromosome arm 5BL, which acts as a chaperone, preventing promiscuous homoeologous pairing to similar, but not homologous chromosomes of the wild/weedy species. Thus recombination must occur during backcrossing after the wheat Ph 1 gene has been eliminated. Based on these findings, we speculate that Ph 1 could be used to prevent gene introgression into weedy relatives. We propose two methods to prevent such transgene establishment: (1) link the transgene in proximity to the wheat Ph 1 gene and (2) insert the transgene in tandem with the lethal barnase on any chromosome arm other than 5BL, and insert barstar , which suppresses barnase on chromosome arm 5BL in proximity to Ph 1. The presence of Ph 1 in backcross plants containing 5BL will prevent the homoeologous establishment of barnase coupled to the desired transgene in the wild population. 5BL itself will be eliminated during repeated backcrossing to the wild parent, and progeny bearing the desired transgene in tandem with barnase but without the Ph 1- barstar complex will die.

Petroleum dependency is a challenge that can potentially be partly offset by agricultural production of biofuels, while decreasing net, nonrenewable carbon dioxide output. Plants have not been domesticated for modern biofuel production, and the quickest, most efficient, and often, the only way to convert plants to biofuel feedstocks is biotechnologically. First generation biofuel feedstock sources: sugarcane and cereal grains to produce bioethanol and biobutanol and oilseeds to produce biodiesel compete directly with needs for world food security. The heavy use of oilseed rape releases quantities of methyl bromide to the atmosphere, which can be prevented by gene suppression. Second generation bioethanolic/biobutanolic biofuels will come from cultivated lignocellulosic crops or straw wastes. These presently require heat and acid to remove lignin, which could be partially replaced by transgenically reducing or modifying lignin content and upregulating cellulose biosynthesis. Non-precipitable silicon emissions from burning could be reduced by transgenically modulating silicon content. The shrubby Jatropha and castor beans should have highly toxic protein components transgenically removed from their meal, cancer potentiating diterpenes removed from the oils, and allergens from the pollen, before extensive cultivation. Algae and cyanobacteria for third generation biodiesel need transgenic manipulation to deal with "weeds", light penetration, photoinhibition, carbon assimilation, etc. The possibilities of producing fourth generation biohydrogen and bioelectricity using photosynthetic mechanisms are being explored. There seem to be no health or environmental impact study requirements when the undomesticated biofuel crops are grown, yet there are illogically stringent requirements should they transgenically be rendered less toxic and more efficient as biofuel crops. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Genes may introgress from transgenic crops into sexually compatible wild relatives via pollen flow. This could potentially enhance the ecological expansion of the introgressed hybrids and their progeny at the cost of other plant species, or affect health of humans and animals, depending on the novel trait engineered into the crop. To prevent generating such competitive transgenic progeny, we previously used tobacco (Nicotiana tabacum L.) as a model for validating a transgenic mitigation (TM) mechanism 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 when in competition with the wild type. In the present study, attempts were made to achieve interspecific sexual hybridization between transgenic TM allotetraploid N. tabacum (pollen donor, representing a crop bearing novel traits) into one of its progenitors, diploid wild type Nicotiana sylvestris (representing a wild relative as well as a progenitor). N. sylvestris plants were manually pollinated by transgenic tobacco. The F1 interspecific sexual hybrids had >75% pollen sterility and produced no seeds. When the F1 was backcrossed as the pollen donor to N. sylvestris, the progeny produced almost no germinable seeds. With such low risk of gene flow, transgenic tobacco bearing novel traits could be cultivated with minimal concern where N. sylvestris is a native or ornamental species.

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.

Models related to plant protection can give an appreciation of a phenomenon, and provide ideas for priorities in research, as well as suggest management strategies. The problem with models is that mistakes can be huge when inaccurate assumptions are made about key parameters, as described with three sets of models: (1) our own model predicting that five herbicide-resistant Striga plants would appear per hectare per season was based on an inaccurate assumption that heterozygotes would be selected, and a heterozygous mutation frequency was used, while a recessive mutant frequency should have been used. A revised model with a recessive mutation would predict five resistant plants per million hectares per season; (2) the model predicting that Bt resistant insects would quickly evolve in transgenic cotton and maize unless massive refuges were instituted, assuming a single binding site for the toxin and minor unfitness of resistant individuals, not realizing that resistant individuals may be extremely unfit or that Bt may have multiple targets; (3) a model that claims that unfit transgenes from crops would decimate wild relatives by swamping. The model assumed animal-type low progeny numbers, and did not consider competition for replacement, nor the infrequency that crop pollen could reach wild relatives. Models must be retrospectively critiqued, based on field data and new knowledge, and not be allowed to become accepted as being axiomatic.

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.

Mechanisms are needed to prevent gene flow from transgenic crops, and the later establishment of these transgenes in populations of other varieties, weeds, or wild relatives. Such prevention can be achieved by containing the transgene within a crop, and then mitigating the effects of the inherent leakage and unidirectionality of containment systems. Mitigation lowers the fitness of recipients below that of the wild-type so that transgenes cannot spread. Transplastomic and male-sterility systems suppress transgene outflow, but not the influx of pollen from relatives, requiring mitigation. The Arabidopsis thaliana Δgai (gibberellic acid–insensitive) gene, driven by its own promoter, induced male sterility in transgenic tobacco (Nicotiana tabacum), which is chemically reversible by kinetin applications. Female reproduction was not affected. Kinetin-treated sterile hemizygous and homozygous dwarf tobacco produced viable pollen, becoming self-fertile with copious viable seed, restoring the small amount of seed production needed for such a crop. Thus, Δgai, under its endogenous promoter, can be used as a containment mechanism to prevent transgene outflow. This application is in addition to the previously described highly effective role of Δgai as a dwarfing mitigator gene, which renders the rare transgenic tobacco hybrids unfit and unable to compete with the wild-type in the mixed cultures. Δgai is unique in that it can be used both to prevent transgene outflow and to mitigate the flow should containment fail or should gene influx occur, a dual role for the gene, not previously reported.

The effects of nanomolar to micromolar concentrations of the herbicide norflurazon were studied in Dunaliella bardawil Ben-Amotz et Avron, a β-carotene-accumulating halotolerant alga. The large amount of β-carotene which Dunaliella bardawil can contain, around 8% of the algal dry weight, is reduced to 0.2% by treatment with 100 nm norflurazon. Simultaneously, phytoene is accumulated to a similar level of about 8%. The gradual increase in phytoene content, in response to increasing norflurazon concentrations, corresponds to the decrease in β-carotene, with no evident change in other isoprenoid intermediates. Carotene-rich Dunaliella bardawil is substantially resistant to high-intensity photoinhibition. This resistance is lost in cells grown to contain low β-carotene and in the norflurazon-treated phytoene-rich cells. These observations are in agreement with the hypothesis that the accumulated β-carotene in Dunaliella bardawil protects the cells against injury by excessive irradiation.

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.

The input costs of pesticides to control biotic constraints are often prohibitive to the subsistence farmers of Africa and seed based solutions to biotic stresses are more appropriate. Plant breeding has been highly successful in dealing with many pest problems in Africa, especially diseases, but is limited to the genes available within the crop genome. Years of breeding and studying cultural practices have not always been successful in alleviating many problems that biotechnology may be able to solve. We pinpoint the major intractable regional problems as: (1) weeds: parasitic weeds (Striga and Orobanche spp.) throughout Africa; grass weeds of wheat (Bromus and Lolium) intractable to herbicides in North Africa; (2) insect and diseases: stem borers and post-harvest grain weevils in sub-Saharan Africa; Bemesia tabaci (white fly) as the vector of the tomato leaf curl virus complex on vegetable crops in North Africa; and (3) the mycotoxins: fumonisins and aflatoxins in stored grains. Abiotic stresses may exacerbate many of these problems, and biotechnological alleviations of abiotic stress could partially allay some predicaments. Some of these constraints are already under study using biotechnological procedures, but others may require longer-term research and development to alleviate the problems. Despite the huge impacts of post-harvest weevils and of mycotoxins in grains, these issues had not been given high priority in national biotechnological programs, possibly due to a lack of knowledge of their immensity. The need for public sector involvement is accentuated for cases where immediate profits are not perceived (e.g. lowering mycotoxin levels in farmer utilized grain, which does not increase yield) but where the public weal will gain, and will be invaluable, especially where the private sector supplies genes already isolated.

Approximately half of aboveground biomass of grain crops (straw) is wasted, with negative ecological and economic impact. Straw use could increase animal production by at least one third if lignin could be transgenically decreased or modified, rendering more carbohydrate available to ruminant animals. Such digestible straw can be upgraded by ammonification, which further separates lignin and serves as a nitrogen source for ruminant bacteria, or biotreatment with ligninolytic fungi and/or physical treatments. Such technologies could supply animal protein to an increasing population, while releasing more grain for direct human consumption without the need to put more land under plough.