**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 [104]. 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 [16] and the Clearfield-Plus\_system [105] 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 [106] or seed mutagenesis [105]. Also, ExpresSun system has been developed as result of mutagenesis breeding [107] with the aim to grow sunflower hybrids resistant to tribenuron-methyl [17].

pollen mediated gene flow in Italy and showed that if time of flowering differs from 4 to 5 days the cross-pollination is reduced by 25%, while difference of 6 days provides 50% reduction [95]. Also, temporal separation in sowing days improves the coexistence of maize [96]. Contrary to that, temporal isolation based on selection of hybrid varieties in which flowering noncoincide achieves the same results although sowing date was the same [97]. Temporal separation and

*Protective vegetation barriers*, made up of one or more different species, can prevent the gene flow by physically stopping pollen in the case of wind pollination. The sowing of conventional crops of the same species, also known as pollen traps, in the vicinity of HR hybrids is an efficient measure, as their role is to attract pollinating insects in order to leave pollen on these pollen traps. This type of barrier can be much more effective than isolation distance. Namely, the sowing of pollen traps between GM and conventional crops is the most efficient measure for the prevention of gene flow [98]. Also, gene flow through pollen from the HR oilseed rape decreases rapidly with the increase of pollen source distance, with the added necessity of a protective vegetation barrier or pollen traps [99]. Some authors [95] studied pollen-mediated gene flow between GM and non-GM maize and concluded that effect of two maize rows surrounding the recipient field in reduction of cross-fertilization is the same like effect of 12

The use of *biological barriers* achieves the best results in the prevention of gene flow, and so far the barriers based on cytoplasmatic male sterility, maternal inheritance, and seed sterility have mostly been used. Cytoplasmic male sterility is based on the inability of plants to produce viable pollen. This type of barrier is suitable option to reduce gene flow in sunflower and maize [43, 66, 100]. Maternal inheritance is successfully used in the prevention of gene flow across the pollen, in the case of several species, including tobacco and tomato [101, 102]. The control of embryo and seed fertility is known as GURT (Gene Use Restriction Technology), i.e., terminator technology, which is considered to be a better control measure, in comparison with sterile pollen production. However, this strategy is seen as the most controversial control measure for limiting genes flow. Additionally, strategies, which include apomixis (vegetative reproduction and asexual seed formation), cleistogamy (self-fertilization without the opening of flowers), genome incompatibility, chemical induction/deletion, etc., are also used in limiting the gene flow [103]. None of these strategies can be applied in all crops, therefore using combinations of different approaches for the prevention of unwanted gene flow is recommended. 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

**5. Gene flow from herbicide-resistance sunflower to wild or weedy** 

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

isolation distance together can be a good solution to minimize unwanted gene flow.

maize rows surrounding the pollen donor.

50 Herbicide Resistance in Weeds and Crops

solution.

**sunflower**

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 [106]. The produced hybrid has shown a high level of resistance toward imazethapyr [108] and imazamox [109], not only regarding different vegetative parameters, but also considering the activity of ALS enzymes *in vivo*, and *in vitro*. Source populations SURES-1 and SURES-2 were used as a source of genes responsible for the resistance to tribenuron-methyl [1, 110], producing also a hybrid with a highly distinguished resistance for this herbicide [109, 111]. The introduction of such crops in the production in Serbian fields has enabled a more efficient control of economic harmful weed species, such as *Sorghum halepense*, *A. trifida*, *A. artemisiifolia*, *C. arvense*, *X. strumarium* and weedy forms of *Helianthus annuus*, their cultivation is also linked with a very high risk of herbicide-resistance gene flow, from these hybrids onto the weedy form of *H. annuus*. Although the presence of four species from the genus *Helianthus* (*H. annuus*, *Helianthus tuberosus*, *Helianthus decapetalus*, *Helianthus scaberimus*) has been confirmed for Serbia, in both crop fields and nonarable lands [112], weedy populations of *H. annuus* occupy the biggest areas, which according to some estimates reach up to 1000 ha in Southern Srem and around 7–8000 ha in Southern Banat [113]. The origin of these populations is not known, but it is possible to determine. For example, origin of French and Spanish weedy populations was determined based on molecular analysis, which has shown that these populations originated from the unintentional introduction of crop-wild hybrids through contaminated seed lots [114]. Difficult eradication of weedy populations due to a high population variability [113, 115, 116] and pronounced invasibility caused by strong vegetative and generative potential [117, 118] presents an additional problem. Besides a reduced sensitivity of this species to nicosulfuron, which is often used as a weed control measure in maize fields where weedy sunflower is present in high densities, has also been detected [119, 120]. Therefore, even though the research into the transfer of HR genes from HR sunflower hybrids to weedy sunflower is in initial stages in Serbia [42, 121], there is high potential for its risk.

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 [122], new crop sunflower varieties are about 65% autogamous [123] 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 [124]. Also, it has been known that there are inter- and intraspecific hybridization between *H. annuus* and its close relatives including its related species [41, 64, 74, 125] or its volunteer plants [65, 69]. In case of gene flow between cultivated sunflower and *Helianthus petiolaris*, the proportion of crop-weed hybrids in the F1 generation varies between 0.3 and 0.5%, depending on flowering period and the presence of common pollinators [68], while in case of gene flow between cultivated and wild sunflower, it was reached as high as 33% [69].

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 [66]. 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 [64] 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 [89]. 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 [43]. 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 [47], while the other [77] 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 [75]. Although, crop hybridization can reduce dormancy in a wild species, hybridization IMI-resistant hybrid and wild sunflower in Argentina did not alter seed dormancy [41], while F1 germination was greater in wild sunflower populations [126].

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.
