*1.7.3. The use of molecular techniques in sunflower breeding for resistance to abiotic stress*

by two genes, a major gene having a semidominant type of gene action (*Imr1*) and a second

Resistance in sunflower can only be achieved with homozygosity (*Imr1 Imr1, Imr2 Imr2*) of

Sala *et al.* [39] reported having obtained a new source of IMI resistance, CLHA-PLUS, devel‐ oped by means of induced mutations. The line was obtained through ethyl methanesulfonate mutagenesis and selection for the herbicide imazapyr. Also, the authors proved at the molecular level that CLHA-PLUS is different from *Imr1* and that both of them are allelic

It has been shown experimentally that the gene CHLA-PLUS has a higher degree of IMI resistance than the gene *Imr 1 Imr 2*. Breeding centers wishing to use the CHLA-PLUS gene for breeding purposes have to sign a contract on its use with the company BASF. At the same time, BASF provides a protocol for screening for resistance at the molecular level (CLEARFIELD®

The recently established CLEARFIELD® (a BASF trademark) Production System for Sunflower provides growers with a new technology, which ensures broad-spectrum postemergence grass and broad-leaved weed control combined with high-performing sunflower hybrids from

BASF Corp. has also established two testing systems which serve to approve IMI-resistant sunflower hybrids as CLEARFIELD®, based mainly on relative tolerance compared with a

Over the last 5 years, there has been a rapid spread of IMI (CLEARFIELD®)-resistant hybrids in the USA, Argentina, and especially central and eastern Europe, where new races of broom‐

Simultaneously with sunflower breeding for IMI resistance, work has been started on the development of hybrids resistant to herbicides from the tribenuron-methyl group of sulfony‐

The first one was derived from SU-resistant wild *Helianthus annuus* plants collected from the same area in Kansas where IMI resistance was found. The USDA-ARS (NDSU) research group incorporated this genetic resistance into cultivated sunflower and released public lines SURES

At the same time, sunflower breeders in various breeding centers (public and private) in the world introduced the sulfonylurea resistance gene into their elite lines, and thus created

The second SU resistance was detected by DuPont within an artificial mutagenesis project conducted in the early 1990s. This material was reselected, purified, and tested by Pioneer/

standard resistant hybrid: Global and Country Qualification System.

*1.7.2. Development of hybrids resistant to sulfonylurea (tribenuron-methyl)*

lureas. To date, two resistance sources have been discovered:

rape, which can be successfully controlled by this technology, have emerged.

gene (*Imr*2) with a modifier effect when the major gene is present.

both resistance genes in inbred line or in a hybrid [38].

596 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

variants of the locus AHASL1 [40].

leading seed companies or public institutions.

Protocol SF30).

in 2001 [41].

resistant hybrids.

Molecular studies as part of sunflower breeding for resistance to abiotic stress should be focused on the recognition of chromosomal segments carrying genes that contribute to the determination of tolerance, provide the possibility to partition the character, and can be used as a tool for an efficient manipulation of the breeding material. For this purpose, genetic maps of neutral molecular markers, such as isozyme and restriction fragment length polymorphism loci, can be an efficient tool for the determination of useful genes [42].

Belhassen *et al.* [43] and Cellier *et al.* [44] were among the first to use molecular techniques in sunflower breeding for resistance to abiotic stress.

Belhassen *et al.* [43] started breeding for drought tolerance from an interspecific cross with *H. argophyllus*. Four cycles of divergent selection using the physiological criterion of leaf cuticular transpiration (relative water loss) allowed the production of two contrasting genotypes: T− (low level of leaf cuticular transpiration) and T+ (high level of leaf cuticular transpiration). Field experiments showed better yield tolerance index combined with good potential yield for T− hybrids in some locations. Physiological analyses conducted in the field and in controlled conditions allowed to distinguish the two genotypes for only one parameter – osmotic adjustment. Molecular comparison revealed the existence of a cDNA differentiating T− from T+. This cDNA has high homology with an amino acid transporter. A quantification of the amino acid concentrations during water deficit in T− and T+ lines showed that the T− plants accumulate significantly more proline than T+ ones. Using this cDNA, RFLP and STS analysis allowed the differentiation of the two lines.

Cellier *et al.* [44] studied a sunflower genotype showing drought tolerance in field conditions (R1 genotype) and another exhibiting drought sensitivity (S1 genotype). They found that R1 tolerance was characterized by a delay of both wilting and decrease of leaf water potential. To analyze R1 tolerance at a molecular level, they isolated different cDNAs (named SDI for Sunflower Drought Induced) corresponding to transcripts accumulated in water-stressed R1 leaves by subtractive hybridization. The analysis of transcript accumulation in both genotypes upon drought stress suggested a differential expression in the *sdi* genes. Abscisic acidmediated induction in the tolerant genotype was observed for four of the *sdi* genes and was found to differ among them. Sequence analysis of SDI clones showed high identity with known proteins, including nonspecific lipid transfer proteins (nsLTPs), early light-inducible proteins (ELIPs), or dehydrin, predicted to be involved in various physiological processes.

Arce *et al.* [45] studied sunflower atypical transcription factors and miRNAs playing a key role in responses to abiotic stresses. In order to achieve the desired results, they used a series of molecular biology techniques. These techniques and strategies include database analysis, phylogenetic tree construction, screening of genomic DNA libraries, isolation of cDNA clones, expression studies using northern blots, western blots, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), functional analyses using plant transfor‐ mation, both stable and transient, confocal microscopy, and microarrays.

Among their findings was the conclusion that transcription factors are proteins able to recognize and bind specific DNA sequences present in the regulatory regions of their target genes. Upon binding, entire signalization cascades are induced or repressed and the plant can adapt itself, at least temporarily, to the adverse conditions to which it is subjected.

Based on the copious results, Arce *et al.* [45] made the following conclusions.

The most amazing results obtained during these studies and other current studies are related to the divergence in structure and function of TFs and miRNAs found in sunflower, apparently conserved in some cases in other *Asteraceae* species but not in model plants. The release of the genomic sequence together with the advance in transformation techniques will certainly help to better understand how sunflower evolved to be adapted to abiotic stress factors and which novel regulating molecules are playing key roles in such an adaptation.

Alberdi *et al.* [46] studied the relationship between a set of molecular markers (amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR)) and leaf expansion parameters under water-deficit conditions in a cross of two public sunflower lines of contrast‐ ing response, in its F2 and F2:3 progenies, and in an independent F8 recombinant inbred line (RIL) population.

Based on phenotypic trials (two in growth chambers – F3 and F2–3) and experiments in a greenhouse (RIL population), certain leaves collected during these experiments were used for DNA extraction. Using a set of 60 SSR and 41 AFLP markers, they achieved significant results, which may be useful for the development of molecular markers for assisted selection in breeding programs oriented to generate new cultivars with improved adaptation to water stress conditions.

Liu and Jan [47] closely studied the results of molecular studies about abiotic stresses in light of their own as well as other authors' research. They concluded that approaches using molecular biology, functional genomics, transcriptome, and proteomics have been used to identify genes or quantitative trait loci (QTLs) and proteins correlated with the network of the response to such stresses, which will provide knowledge for the development of hybrids with resistance or tolerance to them. Some wild species grow in locally extreme environments providing an opportunity to study species from these habitats.

Studying the phenomenon of salt tolerance in sunflower, Lexer *et al.* [48] identified an EST that codes for the Ca-dependent protein kinase with maps to a salt-tolerance QTL in sunflower.

#### **1.8. Conclusions**

Due to the basic structure of its main organs (root, stem, and leaves), sunflower is more resistant to abiotic stresses than other field crops. Therefore, it is usually grown on soils of lower quality ("marginal soils") and in semiarid and arid conditions, where it is often exposed to abiotic stresses.

When it comes to sunflower breeding for resistance to abiotic stresses, the greatest progress has been made in selection for drought resistance. The progress was achieved by using various criteria and parameters, but the most headway was made by using physiological parameters.

The best and the most affordable method for testing sunflower for drought resistance is the use of "stay-green" character. By using "stay-green" in sunflower selection for drought resistance, the selection for *Macrophomina* and *Phomopsis* resistance is made at the same time.

Wild sunflower species of *Helianthus* are successfully used in selection for drought resistance. *Helianthus argophyllus* is most commonly used in selection for drought resistance via interspe‐ cies hybridization. Thus, new germplasms have been developed in a number of breeding centers. Moreover, several more wild species deserve to be used in selection for drought resistance. The use of molecular breeding techniques enables faster and more efficient achievement of desired results in sunflower resistance to drought.

Significant results in sunflower selection for salinity resistance have been obtained by the use of *H. paradoxus* via interspecies hybridization.

Cold resistance can be increased by using certain wild species of sunflower, but especially induced mutations.

Wild species of sunflower are insufficiently used in selection for high temperature resistance, that is, heat resistance, as well as mineral deficiency and mineral toxicity resistance.

By using a population of wild *H. annuus* L. and induced mutations, great headway in sunflower selection for resistance to herbicides from the imidazolinones and sulfonylureas (tribenuronmethyl) group has been made. Sunflower resistance to broomrape (*Orobanche* spp.) has also been achieved.
