Twelfth Australian Weeds Conference

NEEDED: NEW PARADIGMS FOR WEED CONTROL

Jonathan Gressel Plant Sciences, Weizmann Institute of Science, Rehovot, IL 76100, Israel

Proverb: If you never change direction, you will end up where you are heading

Abstract Weeds are the major pest constraint in ex- Molecular biology and biotechnology have much to tensive monoculture or near-monoculture agriculture, offer in upgrading biocontrol, but are hardly utilized as exemplified by the wheat-growing areas of Australia, in inoculum stabilization through to genetic engineer- where a weed such as annual ryegrass (Lolium rigidum ing of increased or decreased host range, increased Gaud) can evolve resistance to virtually every wheat- virulence, or for introducing failsafes. selective and many other herbicides. The evolutionary INTRODUCTION trends towards resistance may have been exacerbated by the penchant for rate cutting that facilitated new Weeds are and have been the major constraint to food types of multifactorial resistances, requiring new man- production in the world. Staying ahead of Malthus has agement models. Herbicide-resistant wheat can only intensified agriculture, increasing practices such as be helpful if the resistance is to a rarely used herbi- monoculture that are questionably sustainable. Sustain- cide, or one not prone to resistance problems, and/or able should be understood in the context of having the resistance is to more than one graminicide, for use in least possible ecological instability. As can be seen in resistance delaying mixtures. The likelihood of herbi- Australia, the evolution of the multiply herbicide re- cide resistant transgenes introgressing from wheat di- sistant biotypes of annual ryegrass is an extreme agonally into closely related grass species is quite low, agroecological perturbation, and current herbicide use and as those species are not weedy, the risk seems very patterns have proved to be unsustainable. long when evaluated with unbiased decision trees. The risk is somewhat higher for oilseed rapid introgressing Similar problems had been happening due to similar genes into related Brassica weeds, where there is a too practices around the world. The heavy use of great a risk. Tandem constructs of herbicide resistance monoculture, typically with the same herbicide regime, genes with “antiweediness” genes could alleviate prob- along with high harvest index, short stature, wimpy lems of introgression of resistance genes from crops (uncompetitive) crops has led to new weed problems. into related species without affecting the crop, e.g. tan- These are not annual or perennial weed problems, these dem constructs with genes that abolish secondary dor- are millennial weed problems. I include amongst them mancy, genes that abolish reactions to shading, or genes Echinochloa spp. in rice and other crops, as well as that prevent seed shattering. Such genes would remain the Striga species in sub-Sahara Africa that are para- tightly linked to the resistance genes. sitic on most crops, halving yields of 100M people (Berner 1995). I also include the grass weeds of wheats Biotechnology can assist weed control in wheat, and such as Lolium spp. around the world, and Phalaris in not just through the development of herbicide-resist- India, that are being mimicked by other grasses in their ant wheat. Modifications of the TAC-TIC (Transposons ability to evolve resistances to graminicides, and not with Armed Cassettes for Targeted Insect Control) necessarily according to mode of herbicide action. This paradigm for insect control using DT’s (deleterious unsustainable instability requires that we analyze why transposons) carrying kev genes (chemically-induced resistance evolved with such a vengeance, and to as- suicide genes) or other deleterious genes would facili- certain whether practices can be modified to delay evo- tate weed control without herbicides. Annual ryegrass lution. would be ideal to test this concept: multicopy transposons bearing desired traits transformed into We must also analyze what biotechnology might have ryegrass could be seeded in pastures. The transposons in store in herbicide-resistant crops and weed would quickly disseminate into indigenous ryegrass biocontrol, and adequately analyze the risks these tech- due to the obligate outcrossing of ryegrass, while not nologies might bring. If the risks of herbicide resist- affecting pastures, but would engender unfitness in ance moving from crops to related species is great, competition with wheat. failsafes should be designed and instituted instead of the two extremes proposed: banning biotechnology, and denigrating or ignoring the risks. Finally, it is posited

462 Twelfth Australian Weeds Conference that biotechnology can be used to reverse evolution; Spraying can be uneven, some weed seedlings are in e.g. to force weeds such as annual ryegrass to evolve the spray shadow of others, weeds such as ryegrass back from being a pernicious weed of wheat to being a often have a second flush of germination when much pasture grass that is not very competitive with wheat of the herbicide has dissipated. The underdosed indi- and disappears when wheat is grown. Some of these viduals are weakened by the amount of herbicide they technologies are unlikely to be developed by industry, receive. Any underdosed, weakened individual, bear- yet are in the long-term public good, and thus the pub- ing a mutation that bears even a small modicum of lic sector with widespread public support will have to survival value may survive and multiply. This muta- take a greater part in such developments. tion could slightly increase the rate the weed degrades the herbicide, could increase the level of herbicide tar- DOSE AND RESISTANCE get, or could slightly protect the weed against the toxic The economics of the extensive cultivation of some- products generated in a sick weed. Many such resist- what marginal agroecosystems in Australia have dic- ance systems are known and they can be governed by tated lowering input costs to the minimum. This has many types of genetic modification: point mutations resulted in the penchant for lowering herbicide rates that slightly modify herbicide binding or slightly in- of application to the bare minimum. Nowhere else are crease the Vmax of degrading enzymes; mutations in such low rates so widely used, which together with the promoters of the herbicide target enzyme or control- biology of annual ryegrass, to my mind explains the ling the levels of herbicide degradation; amplifications problems with that species. in the herbicide target gene; and/or the genes control- ling degradation. Such minor mutations are always When a cut rate that is just lethal to the population “positive” and are thus inherited in a semi-dominant (minimum dose lethal=MLD) is used in the field not manner. The first individuals with the slight modicum all the population gets that dose (Figure 1). of dominant resistance are heterozygous. They will cross with each other and the homozygous individuals with a double dose of the resistance conferring alleles will be selected for under the selection pressure of the low herbicide rate. Any individual bearing a further mutation will be even more viable and will cross with

others. Thus, under low doses the mean LD50 for the population can slowly creep up due to this multifacto- rial mode of resistance, which was loosely termed poly- genic, as population changes are similar to those gov- erned by quantitative inheritance. A pioneering epidemiological study was performed by Figure 1. MLD (minimum lethal dose) in field condi- Ian Heap (1988) in Ron Knight’s lab in Adelaide just tions. Only higher doses provide the MLD to the whole after the first four cases of ryegrass resistance to population in the field, low doses provide the MLD to diclofop were discovered and initially characterized most individuals, Presumed distribution of diclofop on by brother John Heap (Heap and Knight 1982, 1986). annual ryegrass the field at 400, 750, 1200 g ae/ha, Ian Heap collected a large number of ryegrass seed illustrating the proportion of pests receiving each dose. samples from farmers who had used diclofop for dif- Double spraying is ignored, as are untouched escaped ferent periods of time. His data (Fig. 2c) have the typi- organisms (in ‘refuges’). Assumptions: each mutant cal scatter expected from field-derived materials col- gene dose provides protection for 50 g/ha beyond the lected from farmers with limited records on herbicide threshold of 200 g/ha. The cross-hatched area shows use history, but the pattern is clear as well as highly the sensitive population from which one gene dose will significant statistically: there is a creeping increase in be selected at low rates. Reprinted by permission of the mean level of resistance of whole populations that the American Chemical Society; from Gressel (1995) is a function of the number of seasons diclofop was used. This is seen by looking at the population slopes Unlike in the laboratory where precision equipment is that are indicative of mono-modal creep (Heap pers. used to spray synchronous weed populations, field dis- comm), and are not bimodal as intimated by Preston tributions are imperfect. Four-leaf plants are effectively and Roush (1998). This is illustrated schematically as underdosed when the MLD for three leaf plants. population shifts in Fig. 2D.

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Figure 2. “Sudden” appearance of major monogene resistance vs slow incremental creep of quantitatively inher- ited resistance. (A) Actual field data on resistance showing changes in weed populations in a monoculture maize treated annually with atrazine. Amaranthus retroflexus, Echinochloa crus-galli and Digitaria sanguinalis, the foremost weeds, were counted. The maize field was treated with atrazine from 1970 onwards (B). A population distribution description of the same data for Amaranthus in (A), where the relative dose rates (R/S) on the hori- zontal axis are arbitrarily plotted. (C) Slow incremental increase in the dose level of resistance in repeatedly treated Lolium populations. The line showing how the dose required for control may increase was drawn for demonstration purposes only. Lolium rigidum was treated with a typical annual rate of 375 g/ha diclofop-methyl. The relative dose level needed to control resistance in populations is shown as a function of the number of diclofop-methyl treatments. The sensitivity of determination of resistance was lost above a 500 fold increase in relative dose. The populations of seeds were collected in farmer-treated fields and tested by Ian Heap at the Waite Institute. Modified and redrawn from Heap (1988). (D) A population distribution description of the data in (C) where the dose rates on the horizontal axis are arbitrarily plotted. Reprinted by permission of the American Chemical Society from Gressel et al. (1996)

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Unfortunately, no genetic data have been reported on to increase the level of resistance to glyphosate five the inheritance of resistance in ryegrass and there are fold in Lotus corniculatus, a species where biotype still insufficient data on the physiological biochemis- resistance to glyphosate was found well before that in try of the mode(s) of resistance. As predicted >10 years ryegrass (Fig. 3) ago (Gressel 1988) one mode or resistance is clearly Another case of low-level but widespread resistance correlated with a slightly increased diclofop degrada- of a grass to a graminicide is that of Phalaris minor to tion, due to increases in monooxygenase action. isoproturon in India (Malik and Singh 1995). There, Far more is known about a parallel yet less widespread farmers who used full rates, evenly spread over the weed; Alopecurus myosuroides (blackgrass) evolved field, at the right time, without burnt rice straw (=acti- resistance to a wide variety of wheat selective vated charcoal) on the soil surface did not have resist- graminicides under the selection pressure of ance. Those that underdosed by rate cutting, uneven chlorotoluron. This weed also has multiple germina- hand broadcasting of herbicide, late treatment (6 leaf tion flushes during the long growing season of (true) stage), or with burnt straw were the first to have resist- winter wheat in Europe, and the residual herbicide was ance (Gressel et al. 1994). Here too recurrent selec- applied once per season, and soil levels of herbicide tion allowed I50s to creep up (Malik and Singh 1993). slowly decrease. What would have happened if the farmers had used Blackgrass, like annual ryegrass is an obligate intermediate or high rates of diclofop or glyphosate outcrosser and is highly polymorphic and the genetic on ryegrass? From the herbicide distribution curve variations in a large number of populations, with vary- (Fig. 1) it is clear that higher rates assure that all the ing resistance patterns has been exquisitely studied field receives amounts that are above the MLD95 of (Chauvel and Gasquez, 1994). In the “Peldon” resist- individuals who have amassed one or even two gene ant population (which is the most analogous to the doses for resistance, but not enough for controlling multi-herbicide resistant populations of ryegrass), re- weeds containing more gene doses. Thus, two man- sistance is inhereited by “at least” two separate semi- agement elements become clear: (1) evolution of dominant alleles. Two such semi-dominant alleles multifactorially quantitative inheritance will be pre- would be able to allow creeping to four different I50 vented by using intermediate or higher dose rates; (2) levels of resistance. In parallel, but without genetic if one waits until there are too many resistant alleles analysis, the same Peldon blackgrass has been found accumulated to apply an “intermediate” dose, the dose to have two distinct biochemical mechanisms confer- will act like a low dose insofar is it can now select for ring resistance; cytochrome P-450 monooxygenase more such alleles. This means that proactive resistant type of herbicide degradation (Hall et al. 1997; Hyde management strategies must be instituted, not closing et al. 1997) as well as elevated glutathione peroxidase the stable door after resistance predominates. activity that can detoxify the oxygen radicals gener- What happens using only high doses? At continu- ated by the herbicide until it is degraded (Cummins et ally high doses major genes for resistance, i.e. genes al. 1997a, 1999). that with a single mutation confer a huge modicum of Clearly single cytochrome P450 monooxygenases are resistance; often hundreds of fold greater than the wild not the sole cause of ryegrass resistance to diclofop: type are the only type that can be selected. Such muta- single inhibitors of monooxygenase action only par- tions are typically (but not exclusively) in the gene tially suppress resistance. coding for the target protein to which the herbicide binds. In locales where full (high) doses of diclofop Glyphosate resistance in ryegrass has also been found are used on ryegrass such major mutations have been where extremely cut levels were used; 150g/ha on the only type of resistance found in ryegrass. This is young seedlings (Pratley et al. 1996) and 550 g/ha on because you would have to simultaneously have had a older material in orchards (Powles et al. 1998). In- large number of multifactorial alleles for resistance. deed, recurrent selection of the resistant population The likelihood would be low if each were in the popu- resulted in an I shift of the population to a higher 50 lation at a frequency of 10-5, the likelihood of four to- level (J. E. Pratley pers. comm,). The only difference gether would be 10-20, six 10-30, etc. found between resistant and susceptible populations was a doubling of the level of EPSPsynthase activity, the target of glyphosate action (Gruys et al. 1999). A doubled level of EPSPsynthase activity was sufficient

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o be both cost effective, and holds resistance at bay longer than using high or low doses alone. The idea is to use r2 = 0.691 3.0 the MLD for 2-3 years (while only a single allele can accumulate and before a resistance to at least two or 2.5 more alleles can accumulate) and then use an interme- o diate dose that is capable of controlling individuals o o o that have accumulated resistance to at least two alleles, 2.0 o o i.e. probably 600-800 g/ha in the case of diclofop. This o (nmol/min/mg) should remove such individuals and allow the farmer 1.5

EPSP-S specific activity to return to the MLD for a few more seasons. o

50

I

o Such mathematically “proven” models can only be 0.2 0.6 1 1.4 validated by large scale field testing along with care- ful monitoring. The model allows the best of both I50 Glyphosate (kg/ha) worlds, low or intermediate rates, which select more slowly for single major gene inheritance; occasional Figure 3. Relationship between the I for glyphosate 50 intermediate herbicide rates, which should set back any and the specific activity of EPSP synthasses in differ- multifactorial type resistance that may be evolving. ence strains of Lotus corniculatus. (Plotted from data Low/high revolving doses could be used where major in Table 4 of Boerboom et al. 1990) dominant single gene inherited resistance is not ex- pected, as with glyphosate. Multifactoral resistance can evolve while sequential The above interpretation of the evolution of multi-her- accumulation of such alleles is under the pressure of bicide resistance in ryegrass under selection pressure MLD treatment. Initially, major gene mutations for of diclofop is at variance with that of esteemed Aus- diclofop resistance were not found in the resistant tralian colleagues (Preston and Roush 1998). Unfor- populations in Australia, only in North America, where tunately, in the more than a decade of intensive re- only high doses are used. Later they were found in a search on resistant ryegrass, no genetic studies have small population of the resistant populations in Aus- been reported on the mode of diclofop selected multi- tralia. This indicates that there are many more multi- ple resistance and far too little has been published on factorial genes to choose from than the single major biochemistry of resistance. This author feels that Pres- gene. This view is supported by the varying cross re- ton and Roush (1998) were too glib in discounting the sistance patterns for other herbicides found among the large scale epidemiology (Fig. 2c) and they give the ryegrass biotypes, even among the few biotypes that data obtained an impossible alternative possible ex- only saw diclofop in their past history. This indicates planation; that the intermediate I50 levels are due to that each biotype is a mix of different genes, all con- averaging mixed resistant and susceptible populations. ferring diclofop resistance, but having somewhat dif- The statistical data show that this is not the case (Heap ferent abilities to confer resistance to other wheat pers. comm.); there are moving bell shaped curves as graminicides. depicted in Fig. 2D. They also ignore the excellent genetics (Chauvel and Gasquez 1994) and biochemis- Theory (Gressel and Segel 1978), intuition, and some try (Cummins et al. 1997a, 1999; Hall et al. 1997; Hyde practice suggest that the higher the herbicide dose rate, et al. 1997) performed on blackgrass, an analogous the greater the selection pressure eliminating suscep- system. Even less fortunate is that they believe that tible individuals, the more rapid the evolution of pre- their simulations “show” (prove) instead of suggest, dominantly resistant populations. and they confuse simulated data with actual data. Mod- Can you deal with Catch 22 If this analysis is cor- els can at best suggest what field experiments should rect, minimum lethal low doses select for multifacto- be done. Clearly, much more information is needed rial resistance, high doses rapidly select for major gene about resistant ryegrass than is presently available: the resistance, and intermediate doses select slowly for biblical warning to “know thine enemy” has not been major gene resistance. This is a three-way Catch 22. sufficiently heeded. We have intuitively (Gressel et al. 1996) and mathematically (Gardner et al. 1998) modeled a re- volving dose strategy that mathematically “proves” to

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What to do about resistant ryegrass? The area of Canada no longer practices a double standard and all resistant ryegrass in wheat has been increasing for more newly resistant crops must go through the same hur- than a decade – quite a bit since Ron Knight’s first dles, which makes sense if the question is the likeli- cases were discounted as “being unimportant”. In an hood of introgression from crop to weeds. early review of the worldwide situation in wheat, I The needs for BD-HRC Millions of hectares are be- pointed out that wheat has but one biochemical path- ing planted with biotechnologically derived herbicide way to degrade herbicides allowing for selectivity – resistant crops (BD-HRC), mainly in the western hemi- monooxygenases. This is in contrast with the wide sphere and Australia. (The term “genetically modified” spectrum of pathways available in other species is scientifically untenable, as all crops have been ge- (Gressel 1988). The evolution of resistance in ryegrass netically modified.) The farmers perceive the utility to all wheat selective graminicides under the selection of the use for the BD-HRC, as they have many alter- pressure of diclofop suggested a futility in looking for natives and still repeatedly purchase seed of BD-HRC. new graminicides for wheat. The conclusion was that These farmers work on very small margins to mass new selectivities would only come from genetically produce the commodities they grow, and the value of engineering genes conferring resistance to graminicides BD-HRC may be only marginally better than previ- that normally kill wheat (Gressel 1988). The initial ously used cropping practices. Longer-term considera- premise was partially wrong; “protectants” could be tions, which evaluate and support such uses are neces- added to some herbicides that elevated herbicide- sary. degrading glutathione transferases in wheat, confer- ring resistance to herbicides such as fenoxaprop (Tal The real values of BD-HRC come from instances where et al. 1993; Cummins et al. 1997b). The utility of this there really are no viable weed control methods, and novel use of protectants is great, until target site re- the impact of such BD-HRC could be considerable sistance evolves, as it has in many grass weeds. towards a more sustainable world food production. Both the chemical and biotechnological industries have There is a definite need to engineer herbicide not shown particular interest in generating needed BD- resistances into wheat, as both target site and meta- HRC. bolic resistance to graminicides is becoming rampant, throughout the world, not just in Australia and India Two rather different extreme cases where BD-HRC can (Gressel 1998; Heap 1999). make a large impact on Australian production, are briefly presented below. INTROGRESSION OF TRANSGENIC HERBICIDE RESISTANCE FROM CROPS TO Wheat - a crop in need of new resistances Wheat is WEEDS often cultivated where few other cash crops are grown, precluding widespread rotations. The high harvest in- Risk assessment It is clear that in the Australian con- dex, large ear, short-stature varieties are poor competi- text, such transgenic crops have much to offer. From tors with weeds and can rarely be cultivated without the above interpretation of resistance, it is the only hope using cost-effective herbicides. Minimum tillage sys- for wheat, especially continuous monoculture wheat tems requiring more herbicides fit wheat agro- where it is desirable to rotate herbicides. It is also nec- ecosystems with fragile soils. essary when there is a desire to rotate crops. In many agro-ecosystems the rotational crops are left with Grass weeds have evolved morphological and pheno- ryegrass having resistance to a multitude of herbicides, logical mimicries to wheat for 6000 years (Barrett or the rotational crop having hard to solve weed prob- 1993). They have recently evolved biochemical lems of its own. An example is oilseed rape, which has mimicries that overcome wheat herbicides. The muta- both related brassica weeds and ryegrass as problems. tions confer cross resistance to herbicides that are Both genetic engineering as well as other biotechno- chemically unrelated to the selector and have different logical tricks (such as mutation and interspecies modes of action (Gressel 1988; Moss 1992; Powles crosses) have been used to generate herbicide-resist- and Holtum 1994; Malik and Singh 1995; Singh et al. ant crops. 1998a,b). Such resistances cover 40% of Australian wheatlands and millions of hectares in India. Non- Most countries have a scientifically untenable double chemical alternatives raise production costs, severely standard in assessing the hazards from such crops that jeopardizing supplies of the world’s major food grain separates those strains derived obtained genetic engi- and the existence of farming in these areas. neering (even when plant genes are used) from others.

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The immediate answer is to engineer resistances to all moral philosophers (Kline 1991) or environmen- inexpensive herbicides (Gressel 1988). Because too talists (Lewis 1992) share these radical views. little profit is perceived to come from wheat seed or We are warned that these crops can lead to the evolu- even generic herbicides, wheat must be engineered by tion of “superweeds” that will inherit the earth (Kling the public sector. The situation may have changed in 1996). The rapid commercial release of such crops, Australia, where breeders rights have been strength- often without broad-based scientific scrutiny, leads to ened, creating an incentive to create new BD-HR a certain degree of public skepticism about the needs, wheats. Glufosinate resistance has been engineered into utility, risks, and values (beyond profit) associated with wheat, more as a marker gene than for utility (Weeks the use of BD-HRC. The severe pressures of the anti et al. 1993). It is a environmentally and toxicologically groups on policy makers makes it politically incorrect safe herbicide to use, but expensive to manufacture for pursuing public-sector research in this area, which (via dangerous organophosphorus intermediates). affects obtaining accurate information about the dan- Glyphosate resistance has been engineered into wheat gers. These pressures also prevent generating crops and field tested for the niche market of hybrid wheat, needing resistance to herbicides where the where it is less needed. The gene has yet to be made agrochemical or seed industry perceives little profit. available for use in low-input dryland wheat. There The situation is further complicated by well-meaning are two hazards to assess with glyphosate and other scientists who are drawn into the debates, but lack the BD-HR wheats; the risk of ryegrass evolving resist- knowledge to balance the issues. The agronomic needs ance to the herbicide, and the risk of introgression of for, and benefits of BD-HRC, have been widely touted, the gene into wheat-related weeds. If glyphosate re- including in a well-balanced book with sections by sistance is to be engineered into wheat, it should be detractors (Duke 1996). along with (“stacked” with) a second graminicide re- sistance, and herbicide mixtures should always be used. Discussions of BD-HRC have often dealt with the pur- Glyphosate used alone will clearly engender evolution ported environmental risks, but have rarely dealt with of glyphosate resistance (Gressel 1996) and/or a shift the risks from a weed biology/ weed science perspec- in weed spectra towards weeds that have never been tive, yet the major stated risk by the detractors is controlled by glyphosate (Owen 1997). The use of claimed to be the BD-HRC becoming volunteer weeds stacked BD-HR wheat and herbicide mixtures will or introgressing with a wild relative rendering it delay the resistance of ryegrass to glyphosate. The risk weedier; the ‘super-weeds’ of the mass media. Attempts of introgression into wheat-related weeds will be dis- at such an assessment based on weed science was re- cussed later. cently made (Gressel and Rotteveel 1999), using a defined set of uniform criteria set in a decision tree Oilseed rape has become an excellent rotational crop format. Decision trees, by requiring discrete answers for use with wheat in many places where wheat is to sequential, stepped questions, lower the bias in ar- grown. There are many agronomic advantages of ro- riving at conclusions vis a vis the risks deriving from a tating a dicot with a monocot, especially vis a vis given hazard. weeds. It should be far easier to clean up grass weeds in oilseed rape than in wheat as there are more selec- Weeds by definition are very versatile, and have man- tive graminicides for dicots. This is usually correct aged either to evolve resistance, or fill ecological vacu- except in Australia, where ryegrass has successfully ums left by species that became extinct as weeds (cf. evolved resistance to these graminicides. Haas and Streibig 1982). There have been weeds that have evolved resistance to every mechanical, chemi- Ethics, politics and economics of BD-HRC Much cal, or cropping system and management procedure misinformation, disinformation and widely inaccu- agriculture has put in their path. rately-interpreted correct information has been prom- ulgated about BD-HRC (cf. Rissler and Mellon 1995), Conversely, there are wild species that are unlikely ever especially by those with an anti-technology, anti-bio- to become weeds unless they evolve a large number of technology, and/or anti-pesticide bias. Conversely, weedy traits (Keeler et al. 1996). Unfortunately, too those with potential commercial gains from sales of many risk studies do not differentiate between weedy BD-HRC, and/or the increased sales of the herbicides relatives of crops and wild relatives (e.g. Sindel 1997). to be used with them, portray BD-HRC as a risk-free Risk assessment must be performed on a local or re- panacea to agriculture. The detractors often couch their gional basis, as the risks from the same BD-HRC will agenda in political, moral, or environmental terms. Not vary greatly from one agricultural ecosystem to another.

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A second assessment should be done (but has not been terming these special cases as diagonal gene transfer, done in the past), about the effects on weed flora of denoting the grey area where they exist. countries that import bulk unprocessed commodities Vertical and diagonal transfer possibilities are obvi- such as wheat and oilseed rape (Gressel 1997). ous to any plant scientist, but horizontal transfers, with Risks of introgression have been assessed on a case by their disastrous implications to agriculture are not. The case basis. The Canadians were the first to attempt to possibilities of horizontal transfers are extrapolated delineate criteria before even having BD-HRC in place. from the intergeneric and inter-familial plasmid-me- With oilseed rape they first set out criteria “to evaluate diated transfer of traits among microorganisms, which plants with novel traits” (Anonymous 1994a), and then have allowed transfer of antibiotic resistance (a trait specifically evaluated oilseed rape in the context of analogous to herbicide resistance) among unrelated these criteria (Anonymous 1994b). In a series of docu- pathogens. The claim continues that because plasmids ments they further evaluated imidazolinone (Anony- are often used as vectors in the genetic engineering of mous 1995a), glyphosate (Anonymous 1995b), and crops, inter familial transfers will become common- glufosinate (Anonymous 1996) resistant oilseed rapes. place, or at least “inevitable”. This claim does not stand The decision process was based on their perception of up to epidemiological experience with organisms such the risks to regional agricultural ecosystems in west- as Agrobacterium tumefaciens and A. rhizogenes. The ern Canada, and on the scientific knowledge of the plasmids for laboratory gene transfers come from these time. They did not consider other regions (including Agrobacterium spp., which naturally infect a broad the eastern provinces) that may be importing the crops. range of dicots, using the plasmid as part of the infec- Internationally the OECD and UNIDO are developing tion process. If such inter-familial transfer were to oc- a series of “consensus documents” on the biology of cur, it would have been seen over the past 50 years various crops (with regard also to related weeds) so with naturally occurring herbicide resistances. that there is a common starting point to evaluate each There are no known cases where such genes have trans- cropping situation. Their document on oilseed rape ferred inter-familially from any crop to weed via (Anonymous 1997) has been released. Agrobacterium, despite the great selective advantages The source of a gene was an unimportant factor for that such weeds would have. The more than ten mil- Canadian risk analysis. How a gene got there is not as lion hectares of herbicide-resistant weeds that have important as what the gene does in the crop, and how appeared in the past thirty years can all be traced to and whether it will move to weeds, or whether the crop mutational selection evolution and not to plasmid- will become a weed. Much of the stated hazards of mediated horizontal gene transfer. Additionally an ex- interspecific introgression from BD-HRC are based on tensive survey of all the Genebank database found few artificial laboratory experiments, which prove that Agrobacterium DNA sequence pieces in any of the introgressions could happen. Thus they show that the plant genes, which would have been expected in the hazard exists, but give little indication of risk; how millions of years of co-habitation. This matching task quickly such transfers will occur in the field or how fit took hundreds of hours of computer time (Rubin and recipeints will be to cope with competition. The time Levy 1999). Horizontal gene transfer would be a waste factor is not inconsequential; if resistance introgresses of time to discuss further; diagonal gene transfer is a to produce resistant populations more slowly than natu- hazard for which the risks must be estimated. ral mutational evolution, what is the significance of introgression? Generalizing from hazards to risks Because of ge- Two types of gene transfer are widely discussed: (1) netic variability of crops and weeds, and chemical vari- vertical - within a species (2) horizontal - transfer ability in herbicides, their effects and modes of action, among unrelated species, usually by asexual means. one cannot make easy generalizations about the risks Biology is not as clear-cut; there can be some sexual of introgression of resistance. Each case of predicting transfer between species in the same genus and closely the risk of introgression must be evaluated on its mer- related genera that are typically included in some its, often after basic biological, genetic, and epidemio- discussions of horizontal gene transfer. Because ex- logical studies. More importantly, other issues must trapolations are often made from these rare cases of be considered: gene transfer among closely related species to “prove” that all horizontal transfers are possible, we suggest

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(1) What is the benefit to agriculture of having re- homeologous recombination (crossing over between sistance in a certain crop? related but dissimilar chromosomes) would be required when herbicide-resistant durum is used. (2) What are the possibilities of, and implications from, having herbicide resistance pass into a Genes for various traits have been transferred from weedy or wild species? many wild grasses to wheat, especially from the genus Aegilops. Many Aegilops species are considered to be (3) What are the possibilities of and implications Triticum (Kimber and Feldman 1987). The only other from having the BD-HRC becoming a volun- Aegilops spp. known to have a wheat-homologous teer weed in agricultural ecosystems, or become genome is A. squarrosa = Triticum tauschii (Coss.) an alien weed in ruderal or more pristine eco- Schmal. (Kimber and Sears 1987). Only homeologous systems? recombination can occur between hexaploid and The final decision is ultimately a balance between sci- durrum wheats with the other less related species. ence, economics, local benefits, local values, pressure Many intergeneric hybrids generated by breeders do groups, as well as local politics. The politicians often not occur in the field; natural alleles have not passed use science for clearly political decisions (Powell from wheat to wild, related but mainly ruderal spe- 1997). Still, there is good reason that the criteria for cies. The breeders have to resort to forced crosses and risk assessment of BD-HRC should be uniform, using then techniques such as embryo rescue in tissue cul- universal criteria and processes of examination. What ture to save the hybrid embryos that would otherwise is the risk from seeds of a commodity crop for process- abort. ing, when the same seeds would be too risky to sow, yet might escape in the importing country? The onus In Australia, relatedness to wheat is relative. Sindel is on the importing country to demonstrate that it is (1997) in his analysis of crop-related weeds lists none not erecting illegitimate, protectionist, and artificial for wheat. Other Australian weed scientists list trade barriers. Indeed, to prevent trade wars, political Thinopyrum juncerforme, Lophopyrum (Thinopyrum) compromise has led to allowing importation of com- elongatum and Elytrigia (=Agropyon repens as being modities initially claimed to have untenable scientific present in various habitats. The first two are not wide- risks (Powell 1997). “Science” was used for bargain- spread, and rarely in agro-ecosystems, and the last is a ing purposes. All governments should use identical summer weed. Thus, time and place would preclude scientific risk assessment criteria for their mating. As it takes much more than a single gene, even agroecosystems to determine if the risks are much one for herbicide resistance, to turn wild species into a greater in the potential importing country than in the weed (Baker 1974, 1991) the risks of introgression of exporting country. The importer would have to deter- wheat to these weeds are very low. mine whether that the benefits of importing are greater Oilseed rape Most oilseed rapes cultivated are Brassica than the potential costs of mitigating procedures (i.e. napus, a not too ancient tetraploid derived from the eradication of volunteer or introgressed weeds). Indeed CC genome of B. oleracea and the AA genome of B. one could envisage an involvement of the insurance campestris = B. rapa (U 1935). Thus, the only weed industry in risk assessment should there be a require- where homologous recombination can occur is B. ment that importers or exporters insure themselves campestris. Still, homeologous recombination is against such risks. known, especially in the laboratory, with many related Risks of introgression of transgenes to related weeds spp. This required hand pollination after emasculation Wheat Genes from wheat easily introgress into the of the weed, male sterility or self incompatibility in genomes of some related weeds, weeds that are related the weed, massive amounts of crop pollen, and /or to the progenitors of wheat. There is much recent in- embryo rescue of the rare progeny, that are mostly ster- formation of introgression of genes from hexaploid ile or runts (see review of Darmency 1994b; Landbo wheat into Aegilops cylindrica a very problematic weed et al. 1996; Brown and Brown 1996; Mikkelsen et al. in the western plain states and the Pacific northwest of 1996; Metz et al. 1997; Lefol 1996a,b; Bing et al. 1996; the USA (Zemetra et al. 1998; Seefeldt et al. 1998). Scheffler et al. 1995; Conner and Dale 1996, to men- This species shares a D genome with hexaploid wheat tion a few recent papers). Initially, the fear of BD-HRC and homologous recombination occurs naturally un- precluded performing such experiments in the field der field conditions. It would theoretically be much especially where the fears were greatest, in Europe. harder to obtain transfer from tetraploid (durum) wheat This hysteria prevents obtaining field data that might to this species, as durum lacks the D genome. Thus, substantiate, or more likely allay the fears.

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The significance of such laboratory studies to the field highly mutable ALS gene (ca. 10-6 natural resistance situation was evaluated by Karieva et al. (1996). The frequency in populations) (Saari et al. 1994) quickly older epidemiological / apocryphal reports are actu- evolves naturally in weeds. Engineering the same al- ally more relevant to risk analysis than many of the lele with either a two base change coding difference artificial laboratory experiments; the older results could from the natural resistance allele, or with different indicate that such transfers can occur in the field, as introns, would allow easily differentiation of mutational well as the time until predominance, and the competi- events from introgression. This would indicate the rate tive advantage (if any) of such introgressions. of evolution due to introgression vs. the rate of evolu- tion from natural mutation. Most studies rating risks of movement do not differ- entiate between the reports of field transfer and the In the case of triazine resistance, there could be added studies showing it could occur. In the rush to obtain benefits. The trait is maternally inherited, so one might such information, erroneous information has managed assume it will never transfer. Maternal inheritance is to get published in reputable journals. For example not absolute; 0.2% pollen transfer of triazine resist- there was a report of 30% hybridization from transgenic ance was found with genetic markers (Darmency rape seed pollen up to distances of 1 km, >70% in near 1994a). Maternal inheritance of chloroplast-encoded proximity (Skogsmyr 1994). This last number is dou- traits is typical, but there are many cases where such ble the theoretical maximum, as calculated by Connor traits are otherwise inherited (Tilney-Bassett and and Dale (1996). The PCR reactions used to obtain Abdel-Wahab 1979), so it will be necessary to ascer- this fantastic hybridization rate were not controlled by tain frequencies of paternal inheritance with each crop assaying non-transgenic plants, nor were any other situation where maternal inheritance is predominant. biochemical or molecular methods used to verify the Indeed, if the herbicide is used in the BD-HRC, then PCR data, yet this artifactual study is likely to be quoted susceptible weeds growing in its midst will not as fact. Few studies dare to comparatively estimate how introgress the resistant genes; dead weeds don’t have long it will take to have resistance introgress and pre- sex. The genes could introgress into nearby unsprayed dominate in field weed populations vs. how long it weeds. Seed set on emasculated plants from oilseed would take resistance to evolve by natural selection, rape was measured 1.5 km from pollen source vs. the expected commercial lifetime of the herbicide. (Timmons et al. 1996); can the progeny compete, or Assaying introgression in the field There are ways survive in feral populations without selector? With- to ascertain the rapidity of gene movement without out emasculation resistant pollen fertilized 24% of con- causing lasting damage to agriculture and/or the envi- specific plants in the immediate vicinity but <0.017% ronment: simply insert a gene for resistance to a rarely just 10 meters away. If BD-HRC oilseed rape seeds used herbicide. There would be little consequence to carry over as volunteers to other crops where related the herbicide becoming extinct due to the resistance species are serious weeds, gene exchange might hap- disseminating into the wild. Another way to establish pen through this route and long distance dispersal be- the rate of transfer of genes is to abolish the double comes unnecessary. standard; most countries including Australia do not Many weed scientists and others were surprised that scrutinize the use of BD-HRC where the resistance is the Canadian authorities allowed the field use of “natural”, i.e. from selected mutations or artificial ge- glyphosate, glufosinate, (and soon) bromoxynil-engi- netic crosses. ALS-level resistant tissue culture selec- neered transgenic oilseed rapes. The surprise was due tion in crops was acceptable in the USA and elsewhere to the known introgression of herbicide-resistant genes (after registration of the herbicide for those crops). into weeds, including the problematic Brassica Triazine-resistance, laboriously transferred from wild campestris= B. rapa (e.g. Mikkelsen et al. 1996; Brassica campestris into oilseed rape, B. napus Kerlan et al. 1993; Lefol et al. 1996a,b). Brassica (Beversdorf et al. 1980) has been under no special scru- campestris has the dubious distinction of being both tiny in Australia. domesticated to become various crops (Polish oilseed Similar genes introduced transgenically are forbidden rape, turnip, Chinese cabbage, and pak choi) in many in many countries and under scrutiny elsewhere. At places, and the more ancient con-specific wild form is present, if a weed becomes resistant to any ALS herbi- a pernicious weed in other areas (Holm et al. 1997). cides, it cannot be known whether it evolved resist- While botanically identical, they all have different ance naturally by mutation, or was introgressed through morphotypes, with very different phenologies, biolo- cross pollination with a non-transgenic BD-HRC. The gies and competitiveness. Most of the B. campestris

471 Twelfth Australian Weeds Conference crop types are easily controlled. As volunteer weeds using small plots (Scheffler et al. 1995). The authors they have never left agricultural or ruderal areas. Their believe that the frequency would have been even less botanically, but not phenotypically identical weedy twin if larger fields were used, based on bee behavior. The can be very problematic in agro-ecosystems, and was allowable level of cross pollination is 0.1% for “breed- a predominant weed in grains before the advent of se- ers basic” and is 0.3% for “certified” seed, >20 times lective herbicides (Holm et al. 1997). It is hard to pre- more than achieved by transfer at these distances. This dict what will become of the feral populations of Polish indicates that the risk is low when compared to already oilseed rape, which still remain ruderal. They could accepted risks in normal seed multiplication practice; evolve to become more weedy. however far less risk may be acceptable in some cases of potential outcrossing of BD-HRC resistance genes. Deleterious weed genes have introgressed into both oilseed rapes from feral populations, lowering yield The interspecific distaste for alien (non con-specific) and oil quality (e.g. McMullan et al. 1994). A major pollen is a biological barrier to introgression. The use of herbicide-resistant oilseed rapes is to facilitate brassicas are typically considered to be self incompat- control of its wild relatives. The Canadian authorities ible obligate outcrossers. Yet, when solitary B. subjected ALS-R rape derived by mutagenesis to full campestris plants are grown among B. napus, most of regulatory scrutiny before release to the market. Their the offspring on the B. campestris are not hybrids. The decisions (Anonymous 1995a,b, 1996a,b) allow unre- B. campestris plants overcame self-hatred and polli- stricted field cultivation, while noting the likelihood nated themselves, despite ample alien pollen (Landbo of introgression, and stating that the worst case would et al. 1996). In some areas the feral B. campestris flow- be the loss of the particular herbicide to control such ers at a different time as the crop, further diminishing weeds (Anonymous 1995). The decision stated that the mating possibilities. Going further afield to introgression would not increase weediness of crop or intergeneric crosses that have been found in the labo- related weeds outside of agriculture, partly based on ratory, six hybrid seeds were obtained in 50,000 release studies by Crawley et al. (1993). siliques that formed on male sterile B. napus pollinated by Sinapis arvensis (Lefol et al. 1996a). The siliques B. campestris is the weediest of species related to on the supposedly “male sterile” B. napus produced oilseed rape (Holm et al. 1997), and the one species nearly 900 B. napus seeds not hybrids. More impor- with demonstrated field transfer of genes (Mikkelson tant though is the gene flow in the opposite direction, et al. 1996), but not quite as readily as initially pre- from B. napus to S. arvensis. sumed (Landbo et al. 1996). The Canadian authorities did not require two safeguards that might have low- Thus, from all these studies showing that introgression ered the risk of transfer of resistance from B. napus to can occur, we still have little idea how quickly it will weedy B. campestris: occur in field situations, and the only way to learn about the rates of field evolution will be epidemiologically 1. Choice of genomes. If only plants bearing resist- following large scale field use. Still, hybrids between ance on the C genome were used, resistance could only B. napus and wild species are unlikely to perpetuate. transfer to B. campestris by rare homoeologous pair- Farmers will typically cultivate only certified seed; i.e. ing, which seemed to have occurred in an artificial seed from totally weed-free fields, having wide weed- system (Metz et al. 1997). Glyphosate resistance could free areas (200-400 m) around them. Contaminated be coded for by two genes, one for modified target and seed ends up at the crusher, being turned into oil and the other for degrading the herbicide. Resistance trans- meal, without introgressed DNA being perpetuated. fer would be delayed if each were inserted on separate C chromosomes, requiring two independent Many were surprised that the Canadian decisions did homeologous transfers. Whether fortuitously or by in- not call for an active monitoring system to scout out tent, the glufosinate and glyphosate resistances for possible gene or plant movement. Such a system would Canada are on the safer C genome, whereas most of be costly and cumbersome, and for a few years would the resistances studied in Europe that introgressed to probably never find a resistant individual. Still, it would other species were on the B genome (R. K. Downey, only cost ink and the will to print a request on each pers. comm.). seed that a “hot line” be called if patches of putatively- resistant weeds appeared, or if ruderal populations B. napus is self compatible, and is pollinated by in- spread to areas never before colonized. This, together sects. In an isolated field experiment, transgenic traits with a bonded requirement of the commercializers to appeared in <0.02% of seed of non- transgenic oilseed cover the cost of maintaining a hot line and then as- rape planted 200 m away, and in 0.004% at 400 m away certaining whether introgression or movement had

472 Twelfth Australian Weeds Conference occurred, as well as have a liability to eradicate feral a problem, but the crop as a volunteer weed could be- or resistant populations before they spread, would make come a problem, especially if it becomes multiply re- regulatory and biological sense. Much has been writ- sistant to all four herbicides introduced in various va- ten on how comparatively easy it is to eradicate small rieties by cross breeding. There is nothing to stop ge- pest populations, and how impossible it is after they netic engineers from further introducing readily-avail- have reached a critical size (e.g. Moody and Mack able genes conferring resistance to other herbicides 1988; Thill and Mallory-Smith 1997), yet few learn such as 2,4-D (Streber and Willmitzer 1989) in to these lessons about the need for early eradication. oilseed rapes. Nothing would then be left to control B. campestris as a volunteer weed in most Canadian plains Both industry and growers should want to know how crops. quickly and to what extent to expect introgression, and what the consequences might be. As described above, MITIGATING RISKS this could be done cultivating oilseed rape bearing There are various failsafe mechanisms that can be used transgenic ALS resistance and/or bearing transgenic to mitigate the risk of introgression, when and if it does triazine resistance, as it would be possible to distin- occur. guish between volunteer or introgressed offspring from naturally appearing mutants. Apomixis as a failsafe Some apomictic seed is ac- tually of vegetative origin (Koltunow et al. 1995). In the case of ALS resistant oilseed rape, the transgenic Apomixis is being developed to establish hybrid vig- trait would actually be less likely to introgress than our without crosses. If apomictic varieties are pollen the mutant. Publications and patents referring to the free, then their genes cannot introgress into other spe- transgenic oilseed rape state that the 35S promoter was cies or into other varieties of the crop or into con-spe- used. The 35S promoter comes from the cauliflower cific weeds. The lack of viable pollen is probably the mosaic virus (CaMV). The expresson of this promoter only failsafe that would be acceptable to some detrac- is suppressed when herbicide-resistant oilseed rape is tors, who fear intervarietal movement of transgenes, infected by CaMV, and the plants are then sensitive to especially to “organic” crops. the herbicide (Al-Kaff et al. 1997). It has been esti- mated that ca. 80% of Brassica weeds are naturally Gene Placement Failsafes infested with this virus (Cooper and Raybould 1997). Thus most wild Brassica plants that received the gene Chromosomal Wheat and oilseed rape are composed or became feral would still be sensitive to the herbi- of multiple genomes derived from different wild cide, and would have no selective advantage. sources (Kimber and Sears 1997; U 1935). In any given locale it is possible that only one of the genomes of The triazine resistance gene coding an amino acid 264 the crop is identical to that of a related weed allowing transversion from serine to glycine in oilseed rape is easy gene transfer. As the D genome of wheat is com- highly unfit (Gressel and Ben-Sinai 1988). Reciprocal patible with the D genome of Aegilops cylindrica crosses were always less productive with triazine-re- transgenes easily introgress from the B genome of sistant female parents (Beversdorf et al. 1988). They oilseed rape to many brassica weeds and wild species. are grown in Australia (without regulation), where One should perform the cytogenetics to assure that the oilseed rape gene transfer to wild species is consid- transgene is on the incompatible A or B genomes of ered “high risk” (Sindel 1997). The amino acid 264 wheat or the C genome of oilseed rape. transversion of serine to threonine that evolved in po- tato is more fit than serine to glycine (Smeda et al. Hybrids A simple failsafe can be used in hybrid wheat 1993) and should increase yields if transformed into or rape, when and if they become widely commerical. oilseed rape chloroplasts. This should be effective un- If a dominant transgene for herbicide resistance is til B. campestris again evolves resistance to triazines, placed in the male sterile line, there will be no possi- as it had in eastern Canada (Maltais and Bouchard bility of introgression in crop-production areas. Care 1978). will have to be taken in the seed production areas when the male sterile line is restored. Such areas must be The question of how quickly resistance genes will move kept free of related weeds, a typical precaution in seed from B. napus to B. campestris may be moot, as Polish production generally practiced before the advent of oilseed rape (B. campestris) with various herbicide transgenics. resistance genes will soon be released in western Canada (R. K. Downey, pers. comm.). In this region Plastome or chondriome If the transgene for herbi- they do not consider transfer to the con-specific weed cide resistance is placed on the mitochondrial or plas- tid genomes, as has been done in tobacco (Daniell et

473 Twelfth Australian Weeds Conference al. 1998; Kota et al. 1999) there should be little possi- This can well be seen when comparing crops with their bility of gene flow, due to the maternal inheritance of weedy progenitors and relatives (Ling-Hwa and these genomes. Species that are often claimed to have Morishima 1997). no paternal inheritance often have about 0.1% pollen Genetically abolishing secondary dormancy would be transfer of traits. Large scale experiments should be neutral to both crops, but deleterious to the weeds. performed with both crops to assure that the level of Tillage, crop rotation, and preplant use of herbicides, paternal transfer of traits is sufficiently low to justify all standard practices would control the uniformly- using this strategy. There are technical problems in germinating weed seeds lacking secondary dormancy obtaining plastome specific transformation. in rotational crops. Transgenetic Mitigation (TM) Genetic engineering Ripening and shattering Weeds disperse their seed can be used to mitigate any positive effects transgenes over a period of time and much of the ripe seed “shat- may confer. If the herbicide resistance gene engineered ters” to the ground, insuring continuity. A proportion into the crop is flanked on either side by a TM gene in of the weed seed is harvested with crop seed, contami- a tandem construct, the overall effect would be delete- nating crop seed, facilitating weed dispersal to wher- rious to weeds introgressing the construct from a crop ever the crop seed will be grown. (Fig. 4). This is based on three premises: Weeds have evolved morphological and phenological a. Tandem constructs of genes act genetically as tightly- “mimicries” to the crop seed (Barrett 1983; Gould linked genes and their segregation from each other is 1991) necessitating continual evolution and refinement exceedingly rare. of techniques to remove the contaminating weed seed. b. There are traits that are either neutral or positive for Crop varieties have been selected for non-shattering, a crop that would be deleterious to a typical or volun- but recently domesticated crops such as oilseed rape teer weed, or to a wild species. still suffer from shattering (Simon 1994; Prakash 1988; Price et al. 1996). c. Because weeds are strongly competitive amongst themselves, and have large seed outputs, even mildly The first problem in domestication is control of shat- deleterious traits are quickly eliminated from tering (Young 1991; Levy 1985). In addition to the populations. Even if one of the TM alleles mutates, is loss of yield, the shattering of crop seed results in the deleted, or crosses over, the other flanking TM gene becoming a volunteer weed especially in oilseed rape will remain, providing mitigation. (Lutman 1993). Other TM traits that could be used are best visualized Uniform ripening and anti-shattering genes would be when observing the differences between crops and detrimental to weeds, but neutral for wheat (because it weeds. This is best illustrated with two cases: (a) wheat ripens uniformly and does not shatter easily after thou- and weedy relatives (b) oilseed rape (Brassica napus) sands of years of selection), and positive for oilseed and feral and weedy Polish rape/wild radish B. rape, which still has a shattering problem. Crop seed campestris=B. rapa, as summarized below. contaminated with low levels of weed or volunteer seed are typically used for feeding or processing, only weed Seed dormancy Weed seeds typically have secondary free “certified” seed grown where introgression is dormancy with seeds from one harvest germinating bit guarded against is (or should be) sown. by bit throughout the following season, and over a number of years (Vleeshouwers et al. 1995). This evo- Dwarfing. For millenia wheat had been selected for lutionary trait is considered to be a risk-spreading strat- height, to outgrow weeds, limiting the photosynthate egy that maximizes fitness while reducing losses due available for grain. Weed evolution kept apace, giving to sib competition (Hyatt and Evans 1998; Lundberg rise to taller weeds. The advent of selective herbicides et al. 1996). Staggered secondary dormancy prevents to kill weeds allowed for genetic dwarfing of these all the weeds from being controlled by tillage before crops; more seed harvest, less straw. Some of the na- the crop is planted, or controlled by tillage or herbi- tive dwarfing genes were tightly linked to genes re- cides during crop rotation. Rare mutants lacking sec- ducing general yield potential, illustrating the poten- ondary dormancy were selectively propagated during tial for single transgene. Still, the lowering of height, crop domestication, as the loss of secondary dormancy precluding the concomitant problem of tall plants lodg- is desirable to the farmer, who wants uniform germi- ing and increased yield, especially after fertilizer nation after planting the crop. Crop seed that germi- use (which previously promoted lodging), allowed nates uniformly after planting gives a uniform harvest.

474 Twelfth Australian Weeds Conference countries like India to become self sufficient, despite There is considerable debate about the advantages that population increase. would accrue to weeds from the primary transgenic traits. Resistance by modified site of the herbicide bind- Various new systems of genetically engineered height ing to its target should only confer an advantage when reduction are being introduced. These include genes the herbicide is used. It is unknown whether there relating to hormone production (Azpiroz et al. 1998; would be pleiotropic advantages of herbicide resist- Schaller et al. 1998; Peng et al. 1999) as well as those ance due to introducing genes for metabolic inactiva- dealing with shade avoidance. Much of stem elonga- tion of herbicides. tion is in response to shading. This is advantageous when competing with other species, but not in a weed- Let us assume that the primary transgenic trait confers free crop stand where only siblings are competing. The an advantage to a weed; how much will TM traits ac- overexpression of specific phytochrome genes prevents tually mitigate that advantage? Weeds are not only recognition of shading and thus the plant remains short highly competitive with crops, they are competitive (Robson et al. 1996). This is advantageous for a crop with weeds of other species as well as within their own and could also be used where the present dwarfing species. Weeds often produce thousands of seeds, in genes prevent obtaining the highest yields. This trait steady state conditions, to replace a single plant, sug- would be disadvantageous for a weed that must com- gesting extreme competition to be the replacement; the pete with the crops; it would be shaded over by the selection for high competitive fitness is intense. This crop. has dual implications in our situation.

Figure 4. Comparison of current and TM technology. In the current technology, on the rare occasions that transgenic crop pollen (e.g. conferring herbicide resistance, insect resistance, etc.) reaches and fertilizes a live, related weed, there can be some offspring bearing the trait, the proverbial ‘superweed’. In the TM technology, the gene confer- ring the desired trait) is flanked in a tandem construct by TM (transgenetic mitigator) genes that are positive or neutral to the crop, but deleterious to the related weedy species spp. Fertilization of related weedy rices with such pollen will give rise to offspring that are herbicide resistant, but are also non-competitive wimps

475 Twelfth Australian Weeds Conference

After a weed introgresses a transgene and then stabi- Which TM traits are available as gene sequences? lizes (eliminates cytogenetic incompatibilities), the trait Some possible traits for TM constructs just exist as will quickly spread through a population, even if it named genes that are inherited, others are also mapped has a marginally positive fitness advantage (Thill and to positions on various chromosomes, and a few are Mallory-Smith 1997). Conversely, one can balance the actually characterized as sequenced genes. Thus, not disadvantage of TM traits against the advantage of the all TM traits are immediately available for insertion in primary trait. This must be done in both in the pres- tandem constructs. Still, there can be many different ence and absence of the reason for having a herbicide ways for a plant to confer a TM trait, and thus, more resistance trait. Herbicide resistance only provides an than one gene might be available. advantage when the herbicide is used. Indeed, when Secondary dormancy Unfortunately, Arabidopsis, the the herbicide is not present, the transgene resistance typical source for genes, has already been sufficiently trait can be disadvantageous; as demonstrated an ALS domesticated that it is unlike cruciferous weeds; the resistance gene (Bergelson et al. 1996). lab strains no longer have strong secondary dormancy The expression of one gene of a tandem construct could (Van der Schaar et al. 1997). A mutant that is insensi- be lost in the transgenic crop, and sometimes the ex- tive to abscisic acid and lacks secondary dormancy was pression is lost after a few generations. The reasons found in a wild, undomesticated Arabidopsis strain for these losses are not always clear nor relevant for (Steber et al. 1998). Perhaps a way to find more genes this discussion, as only stabilized progeny of is to use the genetic differences between wild transformants are released to agriculture. If all traits Arabidopsis strains and the lab strains presently used, of a tandem construct are expressed after 4-5 genera- as is being done in other instances (Ackerman et al. tions of backcrossing it is fair to consider it stable, i.e. 1997). Much more basic mechanistic research must be as stable as any native, tightly-linked adjacent genes. performed with the crop/weed pairs before the magic ‘abolish secondary dormancy” TM genes can be used. Each TM trait should work in a balance with the pri- mary trait, and where the primary gene gives a strong Shattering Physiologically, one way to avoid seed advantage to a weed, it might be necessary to have shattering is to have uniform ripening. Early maturing more than one TM trait in a construct to obtain bal- seeds of oilseed rape on indeterminate, continuously ance. flowering varieties typically shatter. Determinacy, with its single uniform flush of flowering is one method to The risk of losing TM traits can be further decreased prevent shattering, but this often shortens the season, by combination with a cytogenetic failsafe, where these reducing yield. The hormonology of the abscission are available. If the tandem construct is located on a zone controls whether shattering will occur and it is non-homologous chromosome in wheat or oilseed rape, possible that if cytokinins are overproduced, then shat- then only rare homeologous recombination can move tering will be delayed. As with secondary dormancy, it. As there is no selective advantage to losing the TM no sequenced genes are yet at hand, except for cytoki- trait on the non-homologous chromosome, one can nin overproduction. compound the frequency of likelihood of homeologous recombination with the frequency of loss of the TM Stature limitation Many of the genes used so far to trait(s), reducing risk. obtain vertical deprivation (the politically correct term for dwarfing) seem to have an unknown function. Still The question of how low a risk must we need to attain many genes are known, that control height. is for regulators, but when they do deliberate they must ask; when will the related weed evolve the trait in ques- Gibberellins Preventing the biosyntheses of tion by natural means. The mutations conferring re- gibberellins reduces height (Webb et al. 1998), which sistance to ALS-inhibiting herbicides are naturally is the basis of many chemical dwarfing agents used found in plant populations at a frequency of one in a commercially to lower stature and prevent lodging of million, and ALS-inhibiting herbicides are widely used. wheat. The enzymes and genes controlling various If a TM construct had been inserted in tandem with an steps in gibberellin biosyntheses are known. Copalyl ALS gene, with a likelihood of segregating of 10-10, diphosphate synthase, ent-kaurene synthase, and ent- then the likelihood of getting ALS-resistant weedt rela- kaurene oxidase are responsible for early stages in the tives would not be appreciably changed by biosynthesis of all gibberellins (Smith et al. 1998; introgression from transgenics. Such analyses should Yamaguchi et al. 1998, Hedden and Kamiya 1997; be made wherever possible. Lange 1998; Helliwell et al. 1998). Arabidopsis mu- tations bearing mutations in any of them are dwarfed,

476 Twelfth Australian Weeds Conference with the dwarfing is reversible by gibberellin treatment. situations where there is a strong risk of introgression? Overexpression of a gene coding for ent-kaurene syn- Should such decisions on delay be voluntary? My own thase, causing co-suppression mimicked the mutant view for high risk cases is a paraphrase of a sign about phenotype. Additionally, a gene has recently been iso- playing of radios, seen in the Edinburgh zoo: where lated that confers gibberellin insensitivity when trans- there is a high risk of introgression, “considerate formed into grains (GAI), and thereby induces dwarf- biotechnologists will not release primary genes with- ing (Peng et al. 1999). out tandem TM genes; others may not” release single primary traits. Some processes such as flower stalk bolting are con- trolled by specific gibberellins; in radish GA1 and GA4 FORCING WEEDS TO REVERT TO BEING are responsible (Nishijima et al. 1998). It may be nec- INNOCUOUS WILD SPECIES essary to characterize the genes coding for the The approach of the chemical industry to ryegrass and monooxygenases and dioxygenases that are responsi- the weed problems has been to attempt finding new ble for these later steps (Hedden, 1997). Some of these selective herbicides, while that of the biotechnology genes have been isolated as well (Kusaba et al. 1998). industry to ryegrass and other weed problems has been Brassinosteroids This new group of hormones also generating resistant crops that can withstand herbicides causes elongation of stems in many plant species, and that do yet control these weeds (Gressel, 1998). Agri- their absence results in dwarf plants. A 22 d-hydroxy- culture needs additional paradigms for controlling such lase cytochrome P450 has recently been isolated that weeds. controls a series of these steps in brassinosteroid bio- A group of us propose an added solution, one thought synthesis (Choe et al. 1998), and plants missing the until recently to be in the realm of science fiction: to enzyme are dwarfed (Azpiroz et al. 1998). Addition- genetically debilitate the weed. (J. Gressel E. Rubin, ally, suppressive overexpression of a sterol C24- A. Levy, in manuscript). If this solution is successful, methyl transferase also causes dwarfing (Schaller et it will integrate with and facilitate other successful al. 1998). control mechanisms, leading to more durable control. Shade avoidance Various forms of the pigment phy- Modifying pests to facilitate their control is not novel. tochrome interact to detect whether a plant is being The release of irradiated male insects to impregnate shaded (Smith and Whitelam 1997; Devlin et al. 1998; females with sterile sperm has been used successfully Torii et al. 1998). Phytochrome recognition of shad- in some venues. Here we propose a transgenic ap- ing leads to stem elongation, which is unneeded in a proach, using a wide variety of possible genes that will weed-free crop. The engineering of suppressive be deleterious when turned on; genes that mimic her- overexpression constructs of one of these bicide action; that inhibit plant growth; that render phytochromes led to plants that did not elongate in super susceptibility to herbicides; or modulate hormone response to shading (Robson et al. 1996). Much of the levels. gene isolation has been from Arabidopsis, yet the sup- pressive overexpression was active in dwarfing to- The TAC-TIC approach Pfeifer and Grigliatti (1996, bacco. 1997) proposed a means for controlling pests in two seminal papers called the TAC-TIC model: Because the advantages of transgenic rape are so great, “Transposons with Armed Cassettes for Targeted In- both industry and the farmers are clamoring for sect Control”. In their proposal, an insect is transformed transgenics. They even do so when they know that with a gene, which if activated, can debilitate the in- introgression is imminent. They point out that another sect. They propose to use a chemically-induced pro- resistance will then be available. There are a limited moter to activate genes that would prevent feeding, number of herbicide resistances one can engineer and mating, or otherwise kill the insect. We have termed thus a limited number of times that you can allow re- such chemically-assisted-suicide genes as “kev” sistance to introgress. (Kevorkian) genes. Pfeifer and Griglilatti postulate that Still, much basic and applied research is needed to find not many transgenic pests would be needed if the and use TM genes. Many TM genes may prove to have transgenes are transmitted in a multicopy transposon; near equal worth in increasing agricultural productiv- they will quickly disperse in the indigenous popula- ity as the primary genes presently being used. tion through mating. They suggested that the farmers could use their normal methods of pest control during Should there be a regulatory delay in allowing the the period of transposon dissemination. As no method use of single primary genes, without TM genes in

477 Twelfth Australian Weeds Conference of control kills all pests, the transposon containing and produces double the normal level of EPSPsynthase lacking individuals will be similarly affected, and the (Gruys et al. 1999) as do resistant Lotus corniculatus transposons will disseminate among the remaining strains (Boerboom et al. 1990). A kev gene inhibiting pests – as they would in a population without pesti- EPSP synthase might be less effective on such biotypes. cide. After transposon transmission is complete, the Chemisterilant kev genes. Chemically induced genes use of a chemical that turns on the chemically-induced that will cause pollen sterility a generation hence have promoter replaces the pesticide. been proposed for protecting crop varieties (Masood, Such an approach could be modified and work with a 1998, Crouch 1998, Oliver et al. 1998), the so called weed such as ryegrass, while is solely or predominantly terminator genes of the popular press. Similar con- outcrossing. Indeed, if the proper kev genes and pro- structs could be considered for use as kev genes, when moters can be found, and the transposons available; disseminated by transposons. This could easily be used ryegrass can be easily engineered. Most importantly, in ryegrass planted in pastures, with the terminator gene safety considerations must be dealt with. turned on prior to planting wheat (but not in areas used to propagate seed). This would require always sowing The kev genes can be introduced into a transposon cas- pastures. sette and transformed into ryegrass to generate par- tially debilitated plants after chemical induction, or Debilitating kev genes Ryegrasses have evolved to be possibly before induction. Transposon proliferation highly competitive species, both with the crop as well should enable reaching high copy numbers of the kev as with other weeds and siblings. A typical ryegrass genes in the progeny of these sown parents. The de- plant produces thousands of pollen grains to fertilize bilitating genes will be dominant and high copy one ovule, and thousands of seeds to replace one par- number. Therefore, most, if not all the progeny will ent. Thus, the competition within the species is quite rapidly contain them. This kev containing ryegrass fierce both to fertilize and during the “self thinning” could then be abundantly sown as pasture to increase period when seedlings establish. Some of the genes the frequency of kev-containing plants in the field. proposed for use in TM constructs could be used as DT genes in ryegrass. They would be neutral or posi- The Ac/Ds transposon family, originally found in maize tive in the pasture phase but unfitness that should be (McClintock 1951), has been shown to be active in all rapidly and naturally euthanized from the population. the heterologous plant systems (approx 15 different species) where it has been introduced (See review: Non-chemical strategies would be appropriate for the Kunze 1996). Ac is preferentially transposed during three interbreeding Lolium spp; L. rigidum, L. perenne DNA replication, and as a result its copy number can and L. multiflorum, which are very important pasture increase while it transposes (Greenblatt 1984) grasses in rotation with or near grain fields. There would be nothing wrong with Lolium in pasture phase Potential kev genes In the initial TAC-TIC concept, containing potentially self-lethal kev genes, as long as no specific genes were suggested for use. Perhaps the the chemical inducer is fool proof, i.e. cannot get turned unavailability of appropriate genes (and promoters) on in the pasture phase. An additional or alternative stifled further development of the concept. A plethora approach can be considered with Lolium. One can use of potential genes are available “off the shelf” for use kev genes that are neutral or even positive during the in ryegrass. The various types have different uses in pasture phase but decidedly deleterious during the crop- different systems, but here only those appropriate for ping phase. Thus, kev genes that cause dwarfing by ryegrass are discussed internode shortening or non recognition of shade would One would expect kev constructs to be perfectly use- not matter when cattle represent the only shade as they ful on ryegrass that evolved herbicide resistance by graze the Lolium and its competitors. Genes that se- enhancing metabolic pathways that degrade a multi- verely affect vertical structural stability such as those tude of herbicides. One could envisage inhibiting a joint strongly suppressing lignin formation would also in- promoter region for a plethora of cytochrome P450s crease palatability as well as vastly increase the amount to suppress multiple herbicide resistances, allowing polymeric carbohydrates that can be digested by rumi- control by passé herbicides. Kev constructs might not nant animals. Each percent of lignin in tissue prevents be as effective on weed populations that have evolved the ruminate degradation of three times more cellu- herbicide resistance by overproducing the same target lose and hemicellulose (Jung et al. 1997). Genes sup- gene product as is targetted. It has been claimed that a pressing auxin biosynthesis or stimulating cytokinin glyphosate-resistant ryegrass strain biosynthesis would suppress the dominance of the

478 Twelfth Australian Weeds Conference shoot apex, stimulating branching (tillering) which is The above are general theoretical considerations, but good in pasture but is in competition with crops. Sup- it is clearly necessary to perform a risk analysis based pression of long term (“secondary”) dormancy could on the particular weed, its place in agro- and natural be added in addition to preclude remaining in the seed ecosystems, and whether it has readily introgressing bank for more than one season, insuring that seeds will relations. Horizontal gene transfer among plant spe- not remain overlong in the soil, perhaps even requir- cies has been exceedingly rare since higher plants ap- ing annual storage and sowing. All these genes will be peared on earth. One would consider the same to be beneficial to the pasture species but will render it less true for transposons. One also must consider the po- competitive to a wheat crop, even a very dwarf wheat tential risk from different kev genes under the control crop. of different promoters. One could consider construct- ing decision trees, similar for those constructed to es- Ryegrass has an additional advantage. Whereas few timate the risk of transgenes introgression from crop weeds have been transformed, two species of ryegrass to weed (Gressel and Rotteveel 1999) to deal with such has been shown to be amenable to straightforward issues. transformation techniques (Spangenberg et al. 1995; Ye et al. 1997). Clearly these procedures were eluci- Clearly, despite paper analyss, experiments should be dated because of the importance of ryegrass as a pas- designed to safely evaluate the risk of environmental ture species. hazard (if any) from DT weeds. Chemically induced promoters The TAC-TIC con- Public sector involvement The eventual possibility cept for insects requires the use of chemically-induced of establishing DT containing ryegrass to augment and promoters, although no specific examples were listed partially replace herbicides is different from every day in the initial papers. A wide variety of promoters are agricultural technology where instant (or at least sea- available for chemically inducing the expression of sonal) gratification is achieved. The willingness of the genes in plants (Gatz 1997, Gatz and Lenk 1998). Some pesticide industry to become positively involved is are antibiotics or expensive compounds deemed inap- doubted, because herbicides would be replaced by propriate for agronomic use, but copper salts and etha- cheap generic chemicals used as inducers, and farmer nol are among the inexpensive, simple promoters. profit would be achieved only years after planting DT ryegrass. The biotechnology industry could generate Biosafety of DT The major and worst weeds are not the DT ryegrass, but again there is long delay until wild species. They are truly domesticated, man made profit is achieved. It is just such possibilities where contrivances, just like the crops. In their present evo- the long term benefits for farmer, environment, and lutionary state weeds are not much more competent society have little immediate economic justification than crops to exist in the wild. As with crops, when where the public sector must exert its authority. This ryegrass becomes feral, it can at best exist only in hu- may not be easy in the current climate of demanding man-disturbed (ruderal) ecosystems. Tomes have been that the public sector become increasingly dependent written on how weeds evolved during crop domestica- on the private sector for support. If there is any valid- tion, mimicing crop seed and seedling shape fooling ity to ascertaining whether weeds can be pressured to the farmer. They evolved similar harvest date to crops, become innocuous without pesticides, it can only be ensuring further spread (Barrett, 1983), as well as expected to be performed by public sector research with evolving biochemically mimicry in modes of crop re- public support. sistance to herbicides (Gressel 1988). With DTs, evo- lution is reversed. Most weeds would be forced to re- ACKNOWLEDGMENTS vert back from their highly evolved status in Useful discussions and debates with many colleagues agroecosystems to their status as innocuous wild plants. helped shape the concepts outlined herein. The ability DT constructs would force the evolution of ryegrass to develop these concepts was made possible by the back to being a superior pasture grass, but not a weed. Gilbert de Botton professorial chair in plant sciences. When kev genes are used under a chemical promoter, there should be little danger accruing from the DT transposons entering wild populations of the weed spe- cies or its introgressing relatives. The DT transposon should have little negative value if the chemical in- ducer of the gene is not found in the wild.

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