The insects are exposed to the toxin proteins in transgenic crops throughout the feeding cycle and season, and as a result, the insect populations are under continuous selection pressure. As the toxins are expressed in all parts of the plant, there is a serious threat of rapid development of resistance in insect pests. Toxin production may also decrease over the crop-growing season. Low doses of the toxins eliminate the most sensitive individuals of a population, leaving a population in which resistance can develop much faster.
By far the biggest problem that is likely to occur following the use of transgenic crop plants for pest control is the development of resistance in pest species. This phenomenon, of course, occurs with conventional insecticides. However, the critical difference is that a pesticide application represents a selective force that is time-limited, i.e., to the time the application is made.
The current transgenic crops express toxins throughout their tissues continuously and selective pressure on the target population to adapt will thus be substantial. The strategies for managing insect resistance to toxins expressed in transgenic plants are comparable to those used for managing resistance to conventional insecticides, with only a few exceptions.
1. High Expression Level:
High expression level of toxins in transgenic crops is comparable to the high dose strategy in case of resistance to conventional pesticides. The premise in this case is that toxin concentration is so high that all individuals of the target species are killed. This approach seems to be very effective in theory and practice provided the toxin expression is consistently high and stable over a period of time or among different plant issues.
The high dose strategy assumes that resistance to transgenic plants is recessive and is conferred by a single locus with two alleles, resulting in three insect genotypes, viz. susceptible homozygotes (SS), heterozygotes (RS) and resistant homozygotes (RR).
It also assumes that there will be random mating between resistant and susceptible adults. Under ideal circumstances, only rare RR individuals will survive a high dose produced by the transgenic crop and both SS and RS individuals will be susceptible to the toxin.
The production of toxin is generally expected to decrease over time due to plant senescence. If the level of production of Bt toxin decreases toward the reproductive phase, heterozygous individuals, which may often be slightly more resistant than susceptible homozygotes, might be able to survive and transmit resistance alleles to the offspring.
As Bt transgenic cotton plants matured Bt toxin concentrations decreased, increasing the chances that pests such as Heliothis virescens (Fabricius) will encounter sublethal or low doses of Bt toxins, leading to development of resistance more rapidly. Moreover, different levels of toxins were found in various plant parts, thus increasing the chances that target pests will receive a lower dose.
Similarly, if wild host plants of herbivorous pests or non-transgenic host plants of the same or different species are located in close proximity to the transgenic plants, then pests can move to these non-toxic plants before they receive a lethal dose. Also, an insect displaying a high frequency of movement may feed alternatively on toxic and non-toxic plants thus diluting the dose of the toxin.
In fact, such cases would actually increase the rate of resistance by allowing the survival of partially- resistant genotypes. For protease inhibitors, amylase inhibitors and lectins, the doses that can be engineered into plants are generally insufficient to control a target pest and for these compounds, high dose strategy cannot be implemented. Although some of these substances may be engineered at high doses in the future, many of these are harmful to man, other mammals and birds, particularly at high doses.
2. Refuge Strategy:
Refuges or refugia are areas of crops or host plants free of insecticidal toxins that allow part of the pest population to survive and to act as a reservoir of wild-type susceptible alleles. By maintaining a refuge area close to the transgenic field, surviving individuals (RR) that have been exposed to insecticidal toxins will mate with unselected individuals (SS) coming from the refuge and produce RS heterozygotes that will be killed by the transgenic crop.
This strategy would dilute resistant (R) alleles, reduce the intensity-of selection and delay the evolution of resistance. Spatial organization of refuges has been considered in various ways. One type of spatial refuge includes mixtures of transgenic and non-transgenic plants; the mixture may be obtained by mixing seeds before planting or by planting a smaller proportion of the field with the non-transgenic strain.
Another type of spatial refuge results from the expression of toxins only in critical areas of the plant. A third type of spatial refuge, plantings of Bt-transgenic crops with non-Bt transgenic crops, such as Bt-transgenic corn with non-transgenic cotton, could be applied for resistance management with polyphagous pests such as Helicoverpa zea (Boddie). Temporal refuges involve alternating transgenic plants with non-transgenic plants over growing seasons.
Another aspect of resistance management with refuges is that there must be random mating between individuals from the refuge and the transgenic plants, which means that the refuge must be located close to the transgenic planting. At the same time, for individual pests that tend to feed on several plants rather than just one, the refuge and toxic plant plots must be located far enough apart so that susceptible individuals do not move from non-toxic to toxic plants. Similarly, if refuge fields are less attractive than transgenic plantings to female insects for oviposition, the effective size would be decreased.
According to the recent refuge requirements approved by Environmental Protection Agency (EPA) of USA for Bt corn grown outside cotton-growing areas, growers have to have a minimum of 20 per cent non-Bt corn refuge (treatable with other pest control products). In cotton-growing regions, a minimum of 50 per cent non-Bt corn refuge has to be planted.
Refuges planted as external blocks should either be adjacent or in proximity to the Bt corn field. The current requirement for corn is that the refuges should be within approximately 800 meters of the Bt field, although within approximately 400 meters is the preferred distance.
3. Tissue and Temporal Expression of Toxins:
The tissue and temporal expression of toxins is based on the principle that specific gene promoters could be used to express genes only in- (i) the most important tissue (tissue or structure specific expression) or (ii) critical growth periods (temporal specific expression) or (iii) be environmentally benign chemical.
Thus the production of toxins is limited to the most economically sensitive or most vulnerable parts of plant or to specific time. This strategy does not require external refugia as the plant itself acts as such. However, efficient tissue or time-specific promoters are not yet available.
This strategy is dependent on the feeding behaviour of the pests and can be influenced by the feeding deterrent effect of Bt transgenic plants. Also, an in planta refugia may be relevant for protection against a given member of a group of pests but may fail to provide protection against a secondary pest attacking the plant at the same time but feeding on a different part.
Moreover, several potential economical and sociological problems are related to the use of chemical-inducible promoters. The chemicals to be used for induction might be costly and negatively perceived with respect to environmental protection due to potential pollution hazards.
Tissue-or structure-specific expression could perhaps leave refuges within each plant. This has been suggested for a maize genotype where the Cry toxin is under the control of a phosphoenolpyruvate carboxylase (PEPC) promoter, which provided high levels of expression in green tissue and much lower expression in kernels.
However, the level of expression in the kernel is still 4-6 times the LC50 for Ostrinia nubilalis (Hubner). Damage to corn kernels is tolerable for corn grown for feed, but this level of expression suggests that the ear will not prove to be a very hospitable site for susceptible corn borers.
A slight variation of this idea is to deter insects from feeding on the most economically important plant structures without being killed, thereby lowering selection pressure while protecting the crop. For example, in case of cotton, considerable damage can be tolerated to leaves but not flower buds and bolls.
However, to be fully effective, expression in the transgenic structures may have to be higher than in current constitutive varieties, and will have to provide sufficiently high expression in all important tissues without affecting the tissues that can be sacrificed. In cotton, for example, both bolls and terminal meristems must be protected.
4. Rotations:
In case of transgenic crops, rotations refer to the alteration over time of two or more varieties containing different toxins. Rotation is based on the assumption that the frequency of resistance alleles will decrease when the selection pressure is reduced.
However, many reports indicate that resistance could remain stable or decrease slowly after removal of selection pressure, making the use of rotation inefficient. The resistance of individuals to one toxin must decline during the use of second toxin for rotations-to be effective.
However, assuming no occurrence of cross-resistance between toxins and that resistance does not increase in the absence of toxins, then alternating two toxins over a period of time would actually at least double (if the increase were linear) the time to resistance.
Similarly, alternating three toxins would at least triple the time to resistance, and so on. However, if a fitness cost to resistance or negative cross-resistance exists, rotations can be particularly effective. For insect-resistant transgenic plants, there are limited possibilities for the use of rotations.
Bt toxins have similar modes of action and hence there is a problem of cross resistance, thus rotating between different toxins from year to year is unlikely to slow down resistance. Other insect toxins such as enzyme inhibitors and lectins have not so far given adequate protection when used alone and hence would not be useful in rotations.
5. Gene Pyramiding:
Gene pyramiding or gene stacking refers, to a single crop variety expressing several different toxins, known as pyramiding or gene stacking. This strategy assumes that resistance to each toxin is monogenic, there is no cross-resistance among toxins used, resistant pest individuals are rare so that no one individual is resistant to both toxins and the toxins have equal persistence.
If this is achieved, then the other toxin will kill pests that survive one of the toxins. In contrast to chemical insecticides, at least two features of transgenic plants suggest that mixtures of two or more factors pyramided into the same variety will greatly delay resistance.
Transgenic plants offer a way to get consistently high control of SS homozygotes, and Cry toxins provide at least one good candidate gene with consistently fairly recessive inheritance of resistance. It has been observed that Cry1Aa has overcome a 500-fold resistance to Cry3Aa already established in cotton wood leaf beetle, Chrysomela scripta Fabricius.
Pyramids also have several advantages over single gene strategies:
i. Fitness costs should have a greater impact on delaying resistance to mixtures than single toxins.
ii. Pyramids are less sensitive to initial resistance allele frequency; even resistance frequencies of 10-3 could allow significant benefits.
iii. Pyramids can be greatly improved by manipulating the mortality of susceptible homozygotes, which can be measured.
iv. Pyramids are more robust to survival of heterozygotes, which cannot be measured with certainty until resistance has evolved.
v. Pyramids can be very effective with a smaller refuge.
Ideally, the two toxins to be used in a pyramided variety should be very different to avoid cross-resistance. It is believed that mixtures of plant protease inhibitors may be the best way to prevent insect resistance to these compounds. Plants naturally improve the efficacy of protease inhibitors by exhibiting multidomain or multimeric variants.
Feeding studies have confirmed that inhibitor combinations act synergistically to reduce insect herbivore growth. Since genes for different proteases have been found in nature and engineered into crop plants, the success rates for use of protease and amylase inhibitors in plant resistance to insects can be greatly improved, especially through pyramiding genes that produce a variety of these compounds.
The activity of Bt genes in transgenic plants is enhanced by serine protease inhibitors and tannic acid. Similarly, protease inhibitors engineered into cotton with high gossypol and/or tannic content may achieve greater protection against H. armigera.
6. Mosaics:
Mosaics are a spatial patchwork of two or more varieties of the same crop species expressing different toxins at the same time. For mosaics to delay resistance, it is assumed that there is no cross- resistance among the different toxins, so that the individuals resistant to one toxin are susceptible to other toxins. Thus, susceptible individuals are maintained and are able to move among different patches.
The mosaic theory is not considered appropriate to deploy two toxin genes. It is assumed that each of the varieties can serve as a refuge for the other; however, they cannot since neither is producing many susceptible insects. Even with an untreated refuge, the mosaic system essentially selects for resistance to each of the toxins simultaneously.
7. Trap Plant Strategy:
This approach is closely related to the refuge system. In this approach, the transgenic crop is considered as a trap crop and not as the main source of protection. The Bt-transgenic plants are planted earlier than the non-transgenic plants and owing to their advanced maturation attract the pests that are killed after feeding, thus leaving the non-transgenic varieties relatively unharmed.
There is, however, no evidence that the basic key assumptions on movement and absence of ovipositioning preference will be verified in the field for the target species or for another one. However, a computer model designed on slightly different assumptions about feeding preference and movements demonstrated that this strategy was not successful.
Indeed, avoidance of transgenic plants and ovipositioning preferences were demonstrated. Even if an insect species may be suitable for the trap plant strategy owing to its feeding preferences, the presence of Bt toxins may adversely alter its feeding behaviour.
8. Integration of Tactics:
The most important approach to manage resistance to transgenic plants is to diversify pest management techniques as well as resistance management techniques. This is particularly so in case of multiple crops and multiple pests prevalent in tropical and sub-tropical countries like India.
A toxin may be highly active against a given pest but be moderately active against another pest also present in the same crop or in the same area, and for which the dose delivered by the transgenic plant does not correspond to a high dose. In that case, the second pest will be exposed to a moderate dose and may develop resistance. As there is no single answer or strategy to delay resistance, only a logical combination of various tactics will provide sustainability.
The multiple tactics for Bt resistance management in transgenic crops are as follows:
(i) Provide refuge for susceptibles,
(ii) Do not use pesticides that kill susceptibles,
(iii) Do not use pesticides that kill natural enemies,
(iv) Enhance natural control through crop rotation or tillage, and
(v) Rotate between susceptible and resistant crop genotypes over large areas.
The high-dose refuge strategy, and especially the association of this approach with combinations of toxins is advocated by scientists, seed companies and administrators as the best approach of controlling evolution of resistance. This composite strategy allows flexibility in terms of spatial organization of refuge areas and choice of toxins that will help adaptation and implementation in various agronomic systems.