The majority of the transgenic plants obtained in the previously mentioned publications were developed to establish the transformation methodology with selective markers and reporter genes, like kanamycin resistance gene nptII, phosphinotricin resistance gene bar, glucuronidase gene uidA and green fluorescent protein gene egfp. Nevertheless, there are some studies centred upon the introduction of agronomically useful genes into sunflower.
Three agronomically important transgenic traits were incorporated in sunflower, basically by seed companies: herbicide tolerance, insect resistance and disease (fungal) resistance.
Regarding insect resistance, transgenic expression of the Cry1F (a Bacillus thuringiensis or ―Bt‖ gene) in sunflower confers significant control of Rachiplusia nu and Spilosoma virginica, two important insect pests that impact on sunflower production in various producing countries. A feeding assay was performed using transgenic leaf discs of Cry1F Bt plants and both, larvae at seedling and preflowering stages, clearly exhibited enhanced insect resistance compared to the control. Also, two field bioassays were carried out in Argentina where two Cry1F-transgenic sunflower events exhibited complete resistance to R. nu (Pozzi et al., 2000). Another Bt gene, Cry1Ac, when introduced in a transgenic sunflower line also showed insect resistance. When this transgenic event was crossed with a wild H. annuus accession, the hybrid progeny showed significant enhanced resistance to Lepidopteran damage, which was not a surprising result. However, the transgenic × wild sunflower hybrid produced 55% more seeds than the control hybrids lacking the Cry1Ac gene (Snow et al., 2003). This study confirmed the effectiveness of Bt genes in controlling insect pests in sunflower, and at the same time raised a concern about ―gene flow‖ to wild species that has the potential to enhance the fitness of weeds in the environment.
For fungal diseases like downy mildew and rust that can be controlled by single dominant resistance genes, breeders have been very successful in identifying new resistance alleles in wild species and integrating them into elite germplasm (Gulya et al., 1997). However, for complex fungal diseases like Sclerotinia head rot which is the most damaging disease, the success of conventional breeding was only partial and many breeders feel that the only possible solution is to develop transgenic resistant plants. This approach was carried out by introducing the wheat germin gf2.8 OXO gene (Lane et al., 1991) to generate the so-called OXO-transgenic sunflower plants (Lu et al., 2000, Scelonge et al., 2000). Bioassays of these plants were carried out at second generation (T2) and at more advanced progeny generations indicating the stable inheritance of the transgene. Lesion sizes in the transgenic leaves were inversely related to the endogenous levels of OXO activity and Sclerotinia spread to the capitulum tissue only in control plants while the lesions were confined to the main stem on transgenic plants, proving that OXO can confer significant Sclerotinia resistance in transgenic sunflower (Hu et al., 2003). Moreover hybrids from the crossing of transgenic lines with natural Sclerotinia rot-tolerant lines that carried the OXO transgene were more resistant to Sclerotinia than the corresponding non-transgenic isogenic hybrids, so the combination of OXO with natural tolerance genetic background provides a greater level of resistance than the one observed in the commercial hybrids (Bazzalo et al., 2000). This was the first example of a successful transgene-mediated fungal resistance mechanism in plants. However, the OXO enzyme has been found to persist in simulated gastric fluid digestion studies, raising concerns
about whether the protein may become a human allergen (Jensen-Jarolim et al., 2002).
Regarding the environmental concerns, Burke and Rieseberg (2003) suggest that genetically modified wild plants will not be a worse weed because they do not produce more seeds than the other wild plants, so the OXO transgene will diffuse neutrally after its escape in the absence of selection pressure for Sclerotinia tolerance. Chapman and Burke (2006) studied the escape of transgenes from crop × wild hybridization and showed that natural selection is the main factor governing the spread of favourable transgene alleles, and not the gene flow.
Genetically transformed crops with herbicide resistance genes give farmers greater flexibility for the control of weeds. Among the herbicides used in combination with herbicide tolerant transgenic crops, phosphinothricin and glyphosate have demonstrated effective control of weeds, low toxicity and low impact on the environment. In the case of sunflower, this is also the case for a non-transgenic trait that was introgressed from a wild accession. An imidazolinone (IMI) genetic resistance was identified in a wild population of H. annuus in Kansas by Miller and Al-Khatib (2002).
The basis of this resistance is the expression of two natural alleles containing mutations in the gene that encodes for ALS (acetolactate synthase) that produce a conformational change in the structure of the enzyme which avoids herbicide binding. IMI resistance was incorporated into elite germplasm through conventional breeding and is currently sold worldwide under the ClearfieldTM trademark owned by BASF.
Although this resistance to IMI herbicides was incorporated through conventional breeding methods, some concerns have raised on gene flow of the IMI-resistance trait to wild species. Massinga et al. (2003, 2005) created hybrids of domesticated sunflower with both common sunflower and prairie sunflower with and without the IMI-resistance trait, and measured the growth of IMI-R and IMI-S hybrids under non-competitive conditions, concluding that hybrids of both sunflower species were equally competitive. These results show the safety on environmental impact of IMI technology and besides that the ALS mutant genes can be used for engineering sunflower to produce transgenic sunflower for this or other herbicide resistance genes.
However, this door is not completely open yet, since transgenic tolerance to glyphosate and phosphinotricin have already been introduced in sunflower showing efficacy in field trials, but they did not reach the market because of the concerns regarding the environmental consequences of the transfer of these genes to sexually compatible weedy relatives.
Other transgenic approaches are directed to modify oil quality. Sunflower oil is one of the four major sources of edible oils worldwide and is well known as healthy oil. Besides it is very stable against lipid peroxidation and is used for salad dressings, cooking and frying.
High oleic acid sunflower, Pervenets lines, were originally developed by mutagenesis (Soldatov, 1976). The cross of a Pervenets female with the inbred line HA89 created the first mid oleic or NuSunTM sunflower hybrid (Miller et al., 1987). The existence and the rapid adoption of these lines encourage the improving intentions for oil profile by transgenesis.
The coding sequence of D9-stearoyl-(acyl carrier protein) desaturase from Ricinus communis was introduced into sunflower, under the control of seed-specific promoter and terminator sequences of the late embryogenesis abundant gene from sunflower, Hads10. Fatty acid composition of the seed oil was followed over five generations under greenhouse and in open field conditions.
Some of the transgenic lines produced oil with a significantly reduced stearic acid content compared with non-transformed plants under greenhouse and field conditions (Rousselin et
al., 2002). Dağüstü et al. (2008) introduced into sunflower the Erwinia uredovora phytoene desaturase (crtl) and hydroxymethylglutaryl-CoA (Hmgr-CoA) genes, which have the potential to increase oil quality.
In summary, current scenarios in sunflower transgenesis shows contrasting facts. As mentioned previously, Snow and colleagues (2003) showed some environmental concerns regarding Bt transgenic sunflower release, while Burke and Rieseberg reported lack of such risk after gene flow to weeds for the OXO event.
Besides, as detailed by Cantamutto and Poverene (2007) each new release should be analysed separately as it must be taken into account whether the release would take place in the centre of origin (North America) or in other geographical areas (South America, Europe or Africa) and also that it must be considered the adaptive nature of the introduced character.
For example, it is foreseen that the adaptive impact of a change in oil quality will give no selective advantage to weeds, compared to the case of an herbicide tolerance under the selective pressure of the agroecosystem.
However, even in this extreme example of adaptive advantage it may be argued that it rapidly disappears in the absence of the selective agent (the herbicide) in natural environment outside the agroecosystem.
Moreover, it would be interesting for environmental sustainability to count with the variety of plant tolerant to different herbicides in order to alternate their sowing with other herbicide tolerant species, like soybean or even with IMI sunflowers, thereby preventing the emergence of resistant weeds to one of these herbicides or helping to control them.
As a consequence of the appearance of Clearfield sunflower and its global marketing since 2010, many of the concerns about the release of transgenic sunflowers are rapidly decreasing. In addition, the constant efforts in improving sunflower transformation protocols will strength the emergence of some transgenic varieties of this oilseed in the near future.
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ONCLUSIONThe advances and emergence of new technologies in functional genomics, post genomics and transformation were described in this chapter.
Although sunflower is still an orphan crop, remains as a crop without reference genome available; the development of knowledge at transcriptomics, metablomics and proteomics level along with statistical and bioinformatics procedures for the analyses and data mining of the massive information derived from high-throughput processing methodologies greatly contributes to support plant breeding.
The application of these strategies allows not only the elucidation of complex agronomical pathways but also the identification of candidate robust genes playing key roles in determining these traits.
In addition, the possibility to analyse those candidate sequences as genes or promoters in a transient or stable expression system both in sunflower plants as in model species like Arabidopsis or lettuce assures their functional validation.
These genomics approaches are nowadays being used both in the public and private sectors, and will impact positively on sunflower breeding bringing this crop to a competitive place in the international marketplace.
A
CKNOWLEDGMENTSThis research was supported by ANPCyT/FONCYT, PICT 2011-1365, PME 2007-024, PICT 2007-019, PICT 15-32905, PICT 2007-727, CONICET PIP 5788, INTA-AERG 233261, INTA PE1131024, INTA PE1131023, INTA-PRR AEBIO 241001 and 245001.
SM and GFE are PhD students from CONICET; PF, VL, NBP and RAH are career members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and INTA Researchers; LR and MLB are INTA researchers, HEH is INTA Researcher and Professor at the University of Buenos Aires.
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