Some commercialized HR crops, modified from reference .
Development of herbicide-resistant (HR) crops is way to overcome problems in weed control due to weed resistance to herbicides and absence of new herbicides with a new mode of action for their control. Three types of HR crops were developed: nontransgenic, transgenic, and multiple HR crops. Cultivation of HR crops is associated not only with many benefits (simplification of weed control, more effective and efficient weed control, higher yields, etc.) but also with various risks (development of HR weeds, development of HR volunteer crops, gene flow from HR crops to susceptible relatives, etc.). The greatest risk is gene flow from HR crops to related weed species, wild relatives or conventional crops of the same species. Unwanted gene flow could be prevented or reduced using different barriers such as isolation in space or time, protective vegetation barriers, male sterility, etc. Sunflower hybrids resistant to herbicides (imidazolinones and sulfonilureas) was developed by conventional breeding methods, and their introduction in Serbian fields has enabled a more efficient control of harmful weed species, but the presence of huge populations of weedy sunflower is the main concern associated with their cultivation, because numerous studies have confirmed gene flow from sunflower to its relatives.
- gene flow
- herbicide resistant crops
- wild relatives
The main aim of plant breeding is creating new varieties and hybrids, which would enable us to overcome different problems of contemporary agriculture and achieve high yields and productivity. Research in the fields of molecular genetics, biochemistry, and physiology is leading to development of plants with additional agronomic properties, such as herbicide resistance, pathogen and pest resistance, salt and dryness tolerance, certain food quality parameters, etc. [1–4]. The predominant resistances used in crops are herbicide resistance, both in nontransgenic and in transgenic crops. Owing to the novel insights into the mechanisms and site of action of herbicides on a molecular level, and the development of new biotechnology methods, breeding of herbicide-resistant (HR) crops has been enabled. Thanks to that it is possible to use herbicides, which are preferable from agronomic, environmental, or genetic viewpoint. This new biotechnology gives many benefits in food production such as higher yield through high efficiency of weed control, less unit cost of food production, better quality through removal of existing volunteers of the some species, the possibility of using low-tillage systems, etc. But, this new biotechnology also has some disadvantages such as development herbicide-resistant weed species due to high selection pressure, potential for development of herbicide-resistant volunteer crops, risks of cross-pollination and gene flow from resistant to susceptible relatives, etc.
The focus of this chapter is review of risks associated with HR crops growing with special attention on gene flow from crops to their wild relatives. We first discuss development of HR crops and technologies of weed control based on resistant crops. Also, we briefly discuss gene flow from HR crops to their wild relatives and barriers, which can prevent it. Finally, we discuss transfer of genes responsible for resistance from sunflower hybrids (present resistant crop in Serbia and in Europe) to wild sunflower forms.
2. Herbicide-resistant crops
Discovery of new herbicides, especially with a new mode of action is difficult and expensive. During the last few decades, no one herbicide with novel site of action was found and there are no expectations for its appearance in the near future [5, 6]. One way to overcome this problem was development HR crops, which provide expanding the utility of existing herbicides and improve weed control with them. The study on developing HR crops started soon after the discovery of first herbicide-resistant weeds [7, 8]. These type of crops are designed to tolerate specific broad-spectrum herbicides, which kill the surrounding weeds, but leave the cultivated crop intact. There were two directions in HR crops development, which resulted with two groups of crops: transgenic (genetically modified, GM) and nontransgenic HR crops. The first nontransgenic program for HR crops breeding transferred resistance to herbicide triazines from a
Significant number of crop plants resistant to different ALS (acetolactate synthase; also known as AHAS—acetohydroxyacid synthase) inhibiting herbicides were developed using conventional breeding methods (Table 1). These groups of herbicides have very good characteristics for utilization in weed control in HR crops, which include low use rates, broad spectrum weed control, low mammalian toxicity and environmental compatibility. Immediately after discovery of this group of herbicides, ALS resistant tobacco and maize lines were developed using tissue culture selection [13, 14], while ALS-resistant soybean developed using mutagenesis . After that, three technologies of weed control, which include crop resistance to this group of herbicides, were developed. The Clearfield® and the Clearfield Plus® system have been developed with the aim to grow crops resistant to IMI herbicides , while ExpresSun® system has been developed with the aim to grow sunflower hybrids resistant to tribenuron-methyl . As there is no “alien” genes introduced into these crops, this group of HR crops is not considered as transgenic and has been accepted in countries where the cultivation of GM crops is prohibited , like many European countries, as well as in Serbia.
|Non-transgenic||Photosystem II inhibitors||Soybean||~1991|
|ACCase inhibitor sethoxydim||Maize||1996|
Transgenic (GM) crops developed based on the use of different transgenes, mainly responsible for resistance to glyphosate, which introduced into many crop species (Table 1). These crops became popular thanks to simplification of weed control and reduction of production costs, making the crop more profitable. Between more than a hundred GM products, which have been authorized for commercialization only 13 are crops . The main GM crops are maize, soybean, cotton, and rapeseed, which grow on more than 90 million ha distributed in 14 countries in which these crops have been authorized . These crops are grown in America, Australia, China, South Africa, but distribution is the highest in the USA, where it covers more than 49.8 million ha . In Europe, GM crops (maize, rapeseed, endive, soybean, and flowers) adopted for the production and/or consumption only in few countries, between which Spain is major producer, growing GM maize on more than 100,000 ha .
New approach in development of HR crops is technology, which combines glyphosate resistance with resistance to other herbicides resulting in multiple HR crops (Table 2). This technology developed with the aim to overcome increasing development of multiple HR weeds and based on engineering crops that are able to express multiple HR traits and tolerate multiple herbicides. This new concept using stacked (contains more than one transgene) genes as a tool for postoccurrence and future resistance management is the equivalent to using a single herbicide in case when weed is already resistant to one member of a dual stack . Appropriate transgene stacks should delay resistance longer than approach, which use each component separately and sequentially because each weed resistant to either herbicide will be killed by the other herbicide in the stack. However, that stacking multiple HR into crops may or may not delay the evolution of herbicide resistance because effectiveness of the transgene stacks depends on the management decisions and adoption of the accompanying stewardship programs . Namely, it depends on the effectiveness of each included herbicide in control of each target weed species. Some soybean multiple resistant cultivars have recently been approved for commercial use, such as cultivars resistant to glyphosate, glufosinate, and 2,4-D, as well as resistant to glyphosate and dicamba . Except that it is possible to develop stacks of transgenes for different traits. For example, maize containing transgenes for resistance to insects and to herbicides is commercialized .
|Glyphosate and glufosinate||Soybean, maize, cotton|
|Glyphosate and ALS inhibitors||Soybean, maize|
|Glyphosate, glufosinate and 2,4-D analogs||Soybean, cotton|
|Glyphosate, glufosinate and dicamba||Soybean, cotton|
|Glyphosate, glufosinate and HPPD inhibitors||Soybean, cotton|
|Glyphosate, glufosinate, 2,4-D and ACCase inhibitors||Maize|
3. Benefits and risks associated with growing of herbicide-resistance crops
Cultivation of HR crops is associated not only with numerous benefits but also with various risk factors. The most important benefit is simplification of weed control using herbicides (including nonselective herbicides in many HR crops), in which some crops are able to control weeds that other herbicide that cannot control without concern for crop injury. Also, HR crops are good solution for control of parasitic weed species, in which control is more complex due to their attachment for host (mainly crop) plants . Thanks to flexibility to the time of herbicide application, possible combination with other herbicides and integration with nonchemical methods, this weed control approach made weed management more effective and efficient, which results in higher and more profitable yields. For example, the average increase of yield of glyphosate-resistant soybean in developed countries was 7%, while in developing countries, it was 21% . The higher yield with better quality of seed is not a direct result of HR crop traits
The cultivation of HR crops, whether they have been developed through genetic engineering or classical breeding methods, is fraught with risks, i.e., potential serious economic and ecological consequences. Unlike the HR crops, which have been obtained through conventional breeding methods, the cultivation of GM crops has been a cause of a number of debates, pertaining to the health safety of these products and the risks they present to the environment. The questions, which cause the greatest concern, are those which relate to: (1) direct and indirect toxic effects of products containing transgenes for nonspecific organisms; (2) the impact of modified genes and GM plants on biodiversity, ecosystems, and soil microorganisms; and (3) gene transfer from GM crops to their wild relatives and ecological consequences of this phenomenon . Contrary to this, in the case of HR crops developed by conventional breeding methods, the greatest risk is transfer of genes responsible for resistance from those crops to related weed species, wild relatives, or conventional crops of the same species. Namely, described gene flow creating the hybrids between HR crops and weeds, the so-called “super weeds”, resistant to herbicides. Their eradication subsequently becomes one of the major problems in agriculture. Also, gene flow can change the fitness of recipient biotype/species, whereby increase of fitness resulting in greater weediness, while its decreases lead to extinction . In addition, genes responsible for resistance can flow from HR crops to conventional varieties, which could be the source for resistant genes flow to wild or weedy relatives . Gene flow from transgenic to nontransgenic crops of the same species has been a major controversy, the cause of law suits, and a factor influencing commercialization of some transgenic crops. Some authors  highlighted that the risks associated with transgenic crops cultivation may be more pronounced in the centers of origin of crops than in the other territories because of the presence of wild progenitors and other wild relatives in centers of origin.
The occurrences of volunteer populations of HR crops can also be leading to high risk. Namely, seed dissipation during harvest lead to the appearance of volunteer plants the next season generally in crop production of some crops. Negative consequences of volunteer plants are yield and quality reduction of the crop which they have invaded, contamination of harvested seeds, and maintenance of harmful insects and diseases. In the case of HR crops that volunteer plants basically represent resistant weed populations, which can be a source of pollen, which can contaminate the nonresistant crops or pass the resistance traits onto the related weed species. The control of volunteer plants, which has originated from HR crops, is impossible in the following cultures in which these herbicides are applied as a weed control measure. Therefore, volunteer plants of glyphosate-resistant cotton could be a problem in glyphosate-resistant soybean as subsequent crop  or volunteer glyphosate-resistant canola and wheat could be problem in weed control in conservation tillage system . Also, seeds from volunteer plants of GM crop can contaminate harvest of conventional subsequent crop .
Intensive and repeated use of the same herbicides with the same mode of action in HR crops mainly increase selection pressure on weeds, which would most likely lead to an increase in the selection of HR weed populations. Today, at least 36 weed species have evolved resistance to glyphosate, EPSPS inhibitor (the main herbicide in transgenic HR crops), and at least 159 to ALS-inhibiting herbicides (the main group of herbicides in nontransgenic HR crops) . In addition to these concerns, other negative effects are also possible: herbicide drift can damage conventional crops of the same species, the genes responsible for resistance can be transferred onto conventional crops, characteristics of nontarget plants can be modified, biodiversity may be damaged, and the environment and soil properties can be changed due to the changes in the crop production technologies.
Due to the dangers of the mentioned potential risks, the research into these issues, with the aim of developing suitable prevention strategies, as well as solutions to these problems, should they arise, has been intensified. Consequently, plenty have dealt with the issue of the gene transfer from HR crops to their relatives (wild/weedy forms or conventional crops) [37–42], the study of gene stability in recipients [43, 44], the study of crop-weed hybrid's fitness [41, 45–47] and the competition between crop-weed hybrids and sensitive weed plants of the same species [45, 46, 48].
4. Gene flow from herbicide-resistant crops to wild or weedy relatives
Hybridization and introgression are normal processes, which have continuously occurred between crops and wild or weedy relatives [49, 50], as well as between relative populations of weedy and/or wild species [51, 52]. Even though the hybridization of crops and weeds has an important role in the evolution of many weed species , it can also result in the extinction of certain species related to the crops or the rise of new weed forms, which are more aggressive and better adapted to artificial habitats . There are three types of gene flow:
The ecological consequences of gene transfer from crops to their wild relatives are determined by the quantity of genes, which are being transferred into the populations of wild plants and weeds and the phenotypic characteristics controlled by these genes. Some of the characteristics are insignificant for the fitness of wild relatives, while others (herbicide resistance, disease resistance, and tolerance to the environmental stress factors) mostly improve it. For example, the first generation crop-wild hybrids produced through hybridization between cultivated and wild radish populations [53, 55, 56] was relatively fecund, produced large quantities of seeds and rapidly evolved increased pollen fertility. Contrary to this, if the introduced genes weaken the fitness of their wild relatives, their invisibility will also decrease. This process can be accelerated by introgression and the introduction of new genes from neighboring crops, which ultimately leads to the extinction of the initial populations of wild relatives . Except ecological consequences, gene flow from crops to weedy relatives is associated with many problems in crop production. Namely, the development of HR crops has given rise to the situation where the hybridization is often seen as a problem, particularly when it relates to the hybridization between GM crops and related species. Also, it is important to bear in mind that in some countries coexist different cropping systems, which cultivate conventional, organic, and GM crops. In that situation, there is risk for gene flow between GM and non-GM cultivars through cross-fertilization due to pollen flow between neighboring fields. Progeny of HR crops and weedy/wild relatives or volunteers will be resistant weeds, in which control is difficult.
Genes responsible for crop's herbicide resistance can be spread in the environment as a result of three mechanisms, including gene transfer across a pollen (as a result of allogamy), seeds (as a result of their dispersal) and for perennial species by the vegetative propagules. Potential for pollen-mediated gene flow is higher for both wind and insect pollinated out-crossing crops than for self-pollinated crops . Although gene flow across a pollen is more studied, gene flow by seeds during commerce may be very important for the long-distance dispersal of genes responsible for resistance to herbicides . The both ways of gene flow from HR crops including both GM and conventionally bred HR crops have been confirmed in many cases [37–40, 60, 61].
The transfer of genes from HR crops to their relatives is dependent on multiple factors (Figure 2), such as the coexistence and proximity of the crop and its close relatives, their biology and phenology, type of vector, development of F1 generation, which is fertile and capable of survival, the production of fertile subsequent generations, the potential for gene transmission, chromosome recombination and movement of genes of one species into the genome of another, due to introgressive hybridization and gene persistence in volunteer crop populations [58, 62]. Also, in study about gene flow from glufosinate-resistant rice to improved rice cultivars and weedy rice in China, the conclusion was that gene flow depends on the height of pollen recipient plants . They found that the gene flow was lesser if recipients were taller than in situation when they were shorter.
Cross-pollination between HR crops and sexually compatible wild or conventional cultivated crops of the same species is the major pathway for gene escape. Therefore, transfer of genes responsible for HR between sexually compatible individuals is most often done through pollen, whether within the same population or between different populations [38, 64]. This occurrence is dependent on different factors of which autoincompatibility that enhances allogamy in wild forms, environmental conditions (wind speed and direction, temperature, light intensity, and humidity) as well as the type (wind and/or insect) of pollination vector [37, 38, 65, 66]. In addition to this, the crucial role in gene transfer through pollen lies in the coincidence of the flowering period between the HR crop and its wild relatives. Although experimental data suggest that the flowering period of wild populations is generally longer than the flowering period of crops, which makes the overlap highly likely , in some cases, gene flow between HR crops and relatives was disabled due to flowering period not overlapping or time of overlapping was short. For example, hybridization between imazamox-resistant and weedy sunflower was not confirmed in experiments in Serbia when period of flowering overlapping was short . Also, it was confirmed that the gene transfer from the cultivated onto the wild sunflower in Argentina depended on the overlap between the flowering period and the presence of common pollinators [68, 69]. Pollen dispersal from HR crops onto their wild relatives is also dependent on their mutual distance, the size of populations from which the pollen originates and where it is delivered, plant density, number of flowers per plant, and the location of wild relatives in relation to the crop .
Although numerous studies have confirmed the transfer of genes relevant for HR to their wild relatives, hybridization level mainly was low. Some authors  studied the transfer of genes responsible for imazethapyr-resistance, from the rice cultivars to the weedy rice species in 22 field sites. They confirmed that even though gene transfer occurs, in the majority of sites (18) less than 1% of hybrid progeny was present, while in the remaining four sites that percentage was somewhat higher (up to 3%). Also, low levels of hybridization (1–2%) were confirmed between rice and its wild congener
Despite the fact that the gene transfer from crops to their wild relatives is widely studied, there are no detailed data available on what happens with these genes, which have been introduced into wild populations after a longer period of time. Namely, the majority of this research concludes with the first generation of hybrids. However, genes originating from the cultivated sunflower can persist in wild populations over the five-year period, following the hybridization . Some authors  have also studied the effects of a 40-year long gene transfer from the cultivated to the wild sunflower populations.
Importance of crop-weed hybrids produced as result of gene flow from HR crops to wild or weedy relatives for future crop production can be different depending on traits introduced into progeny. Therefore, assessment of gene flow occurrence requires not only estimating the degree of gene flow, but also evaluating the relative fitness of hybrids. It long dominated the view that crop-wild hybrids have a lower fitness than their wild parent [75, 76]. But, many studies confirmed that some hybrids display increased , while the other display reduced  fitness in comparison with their parents. Displayed fitness depends not only on the crop traits introduced to wild relatives, but also on environmental conditions. Namely, fitness of hybrids between crop and wild sunflower increases in stressful conditions common to conventional agroecosystem like competition and herbicide application .
The role of seeds in the transfer of HR genes from crops to their wild relatives is evident in their spread into new areas where volunteer populations are formed. After that HR genes can be transferred from these volunteer populations to their wild relatives through the pollen. Also, hybrids resulting from spontaneous crosses of HR crops and their wild relatives through seeds can be carried into new areas, where they subsequently present a source of pollen, which carries the resistance genes. Unlike pollen, the seeds usually remain in the close proximity of the plants from which they have originated. But, as seeds are more persistent than pollen, movement of seeds is possible to further distances by human activities then pollen movement . In general, seed dispersal of HR crops or progeny created through their spontaneous crossing with wild relatives, depends on the biological properties of the crop, the ecological conditions, crop production technology and the agrotechnical measures applied on these fields, following with harvest. Nevertheless, it is possible to monitor the dispersal of these seeds in space and time. Some authors  have confirmed the gene transfer of sugar beet to their wild relatives through the seeds whose dispersal resulted from soil transport. Namely, although spontaneous spatial dispersal is often considered as irrelevant since the seeds of a majority of crop cultures have lost this ability, seed dispersal is also possible as a result of spillage during the harvest and their transport and storage operations, which enables the spread to great distances. The dispersal of seeds containing the genes responsible for resistance over time depends on the dormancy characteristics and the seed’s longevity in the soil, as well as the ecological requirements for its germination. Also, it should be considered that, in addition to pollen and seeds, soil seedbank has an important role in the plant dispersal . Namely, when considering different life forms of sugar beet (cultivated, wild, and weedy), it is well known that they form long-term seedbanks , which, over a longer period of time, can provide the plants which are then a source of HR genes.
Gene flow by the vegetative propagules (stolons, rhizomes, roots, crowns, and bulbs) is possible on short distance via natural means or on equipment moved between fields, while long-distance movement could be possible only with human activities or through the waterways . As HR crops are mainly annual species, gene flow via vegetative propagules can be interesting only for perennials like glyphosate-resistant alfalfa (commercially available) and creeping bentgrass (
Pollen flow from crop to the relative seems as relatively simply process, but gene introgression is complex, occurring in several steps which mean several hybrid generations, which can exchange genes among themselves and coexist many years simultaneously (Figure 3). The likelihood of gene transfer from crops to their wild relatives depends on the genetic characteristics of crops and their wild relatives, as well as the homology of their genomes . In the cases where the degree of the homology between the crops and their wild relatives is higher, as in the case of
|Crop||Risk level||Wild relatives for which the introgression of gene has been confirmed|
|Common Bean||Very low|
In order to
The use of
All mentioned measures for prevention and reduction of gene flow are important separately, but their integration and combination with stewardship production system could be the best solution.
5. Gene flow from herbicide-resistance sunflower to wild or weedy sunflower
Options for chemical control of broadleaf weed species, especially weeds belonging to Asteraceae family, without injuring the crop are quite limited in sunflower compared to most other row crops . Due to that, sunflower hybrids resistant to ALS-inhibiting herbicides, including imidazolinone (IMI) and sulfonylurea (SU), was developed by conventional breeding methods, with the aim to improve weed control. The Clearfield_system  and the Clearfield-Plus_system  have been developed with the aim to grow sunflower hybrids resistant to IMI herbicides. For development of those hybrids were used for subsequent crossings between cultivated sunflower and wild resistant sunflower  or seed mutagenesis . Also, ExpresSun system has been developed as result of mutagenesis breeding  with the aim to grow sunflower hybrids resistant to tribenuron-methyl .
The breeding of sunflower hybrids resistant to herbicides belonging to IMI and SU groups in Serbia was started in 2000, and since 2003, this technology has been applied in the production. As a donor of imazamox-resistance gene, the wild sunflower originating from the USA was used, in which the resistance to herbicides of the imidazolinone group was developed following a seven-year consecutive application of imazethapyr . The produced hybrid has shown a high level of resistance toward imazethapyr  and imazamox , not only regarding different vegetative parameters, but also considering the activity of ALS enzymes
The main concern associated with cultivation of HR sunflower is potential gene flow from crop to weedy or wild relatives. Although wild sunflower populations are self-incompatible , new crop sunflower varieties are about 65% autogamous  and weedy population as a result of their hybridization are self-incompatible. Therefore, there is great potential for pollen-mediated gene flow. For example, seed-mediated gene flow from cultivated sunflowers to wild sunflowers may be common . Also, it has been known that there are inter- and intraspecific hybridization between
Gene flow from sunflower crops onto their wild relatives mediated by pollen is dependent on different factors. The overlap of flowering periods of cultivated sunflower and its wild relatives, the pollinators which they share, self-incompatibility of the wild species, diploidy, and high levels of cross-fertilization are all factors which contribute to the spontaneous hybridization . However, the hybridization between the sunflower and its relatives can be absent due to the mismatch of the flowering periods, incompatibility, physical distance, differences in the genetic structure between the species and interspecific competition of pollen [89, 125]. Many studies [42, 70, 121] confirmed that the pollen transfer from the resistant crops to their relatives primarily depends on their distance to the pollen source and the plot size. Consequently, some authors  have confirmed, when studying gene flow from sunflower imidazolinones-resistant hybrids to their wild relatives, that the HR gene was transported to a distance greater than 30 m from the pollen source, while the percentage of the surviving offspring of wild relatives was reduced with the increase in the distance from the HR hybrid. Also, it has been confirmed that the gene flow from the crop sunflower to its wild form is reduced with an increase in their mutual distance, with it being 27% at a 3 m distance. However, gene flow has also been confirmed at a distance of over 1000 m from the pollen sources . Additionally, it was determined that 42% of the wild offspring sunflower at a 3 m distance from the crop sunflower represented its hybrids, while at a distance of 200 m, this percentage was 10%, and 4% at a distance of 400 m . Several authors [42, 45, 64] indicate that the wind direction affects the gene flow, which is ascribed to its influence on the flight of bees.
The main consequence of gene flow between crop and their wild relatives is the increasing of wild relative fitness as a consequence of introgressed genes, which can lead to the development of invasive weeds. Some studies confirmed fitness increase of hybrids between sunflower crop and their relatives , while the other  confirmed hybrids in the first generation after crossing had lower fitness than wild parent in natural habitats, but in the following generations, fitness of hybrid was recovered. Also, hybrids between crop and wild populations of sunflower express lower fertility than their wild counterparts . Although, crop hybridization can reduce dormancy in a wild species, hybridization IMI-resistant hybrid and wild sunflower in Argentina did not alter seed dormancy , while F1 germination was greater in wild sunflower populations .
Strategies for prevention or reduction of gene flow between crop sunflower and its relatives can be developed based on understanding seed and pollen dispersal and influence of different factors on that processes. The biological barriers based on cytoplasmic male sterility, which disable of plants to produce viable pollen, could be good option to reduce gene flow in sunflower.
This work was supported by Ministry of Educattion, Science and Technological Development of Serbia.