*1.2.2. Using different traits in sunflower breeding for drought resistance*

Škorić [7] reported that over 30 different parameters were used in the study of drought resistance and breeding for drought resistance in sunflower. Among these, the most frequently used were physiological parameters.

Chimenti *et al.* [17] reported that high osmotic families extracted more water from the profile during the stress period and had greater grain yield and leaf area duration than families with a low degree of osmotic adjustment. The same authors concluded that osmotic adjustment can contribute to post-anthesis drought tolerance in sunflower through increased water uptake, reduced impact on grain number, grain size, and greater leaf area duration.

Andrei [18] concluded that high self-fertility (24–49%) in some hybrids ensured a greater stability in sunflower yield under stress conditions.

Studying the influence of drought stress on growth, protein expression, and osmolyte accumulation in sunflower, Fulda *et al.* [8] reported that osmolyte analysis revealed an accumulation of glucose (24–30-fold), inositol (20–30-fold), proline (10–20-fold), fructose (3–6 fold), and sucrose (4–4-fold) in extracts from leaves of drought-stressed plants. Changes in protein expression of drought-stressed versus control plants were detected in colloidal Coomasie-stained 2D-polyacrylamide gel electrophoresis (PAGE).

Sato *et al.* [19] studied the correlation between the responses of leaf expansion and hypocotyl elongation to water deficit in sunflower genotypes. Based on the results obtained, they reported that the response of hypocotyl growth to water deficit ranged between 31 and 48%, while that of leaf growth ranged between 40 and 63%. There was a significant positive correlation (*p* < 0.01 *R*<sup>2</sup> = 0.61) between both responses. The correlation was also significant using Pearson's correlation test (*p* < 0.04, *r* = 0.78).

Petcu *et al.* [20] studied physiological traits for the quantification of drought tolerance in sunflower and determined as follows. The reduction in leaf area, shoot size, and biomass accumulation of sunflower seedlings under water stress conditions determined the increase in root/shoot ratio. This suggests that for young plants the main sink was survival. In a late stage of vegetation, the root/shoot ratio decreased under drought stress in some hybrids but increased in others, suggesting that for mature plants the main sink was the yield. The physiology work has focused on morpho-physiological traits induced by drought and associated with drought tolerance of plants and the elaboration of screening methods for rapidly measuring drought tolerance using plants in an early stage of vegetation.

Based on the results of Škorić [7, 11], practical results in sunflower breeding for drought resistance have been achieved by using the stay-green phenomenon. Here, we should warn that in the selection of lines on the basis of stay-green criteria, only lines with a high degree of self-fertility should be looked for, otherwise a wrong choice of genotypes will be made.

Research and characterization of physiological mechanisms in wild sunflower are just beginning. Škorić [16] suggests that in breeding for drought tolerance, there should be a greater effort to expand the use of other wild species such as *H. deserticola, H. hirsutus, H. maximiliani,*

Škorić [7] reported that over 30 different parameters were used in the study of drought resistance and breeding for drought resistance in sunflower. Among these, the most frequently

Chimenti *et al.* [17] reported that high osmotic families extracted more water from the profile during the stress period and had greater grain yield and leaf area duration than families with a low degree of osmotic adjustment. The same authors concluded that osmotic adjustment can contribute to post-anthesis drought tolerance in sunflower through increased water uptake,

Andrei [18] concluded that high self-fertility (24–49%) in some hybrids ensured a greater

Studying the influence of drought stress on growth, protein expression, and osmolyte accumulation in sunflower, Fulda *et al.* [8] reported that osmolyte analysis revealed an accumulation of glucose (24–30-fold), inositol (20–30-fold), proline (10–20-fold), fructose (3–6 fold), and sucrose (4–4-fold) in extracts from leaves of drought-stressed plants. Changes in protein expression of drought-stressed versus control plants were detected in colloidal

Sato *et al.* [19] studied the correlation between the responses of leaf expansion and hypocotyl elongation to water deficit in sunflower genotypes. Based on the results obtained, they reported that the response of hypocotyl growth to water deficit ranged between 31 and 48%, while that of leaf growth ranged between 40 and 63%. There was a significant positive

Petcu *et al.* [20] studied physiological traits for the quantification of drought tolerance in sunflower and determined as follows. The reduction in leaf area, shoot size, and biomass accumulation of sunflower seedlings under water stress conditions determined the increase in root/shoot ratio. This suggests that for young plants the main sink was survival. In a late stage of vegetation, the root/shoot ratio decreased under drought stress in some hybrids but increased in others, suggesting that for mature plants the main sink was the yield. The physiology work has focused on morpho-physiological traits induced by drought and associated with drought tolerance of plants and the elaboration of screening methods for

Based on the results of Škorić [7, 11], practical results in sunflower breeding for drought resistance have been achieved by using the stay-green phenomenon. Here, we should warn

rapidly measuring drought tolerance using plants in an early stage of vegetation.

= 0.61) between both responses. The correlation was also significant

*1.2.2. Using different traits in sunflower breeding for drought resistance*

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

reduced impact on grain number, grain size, and greater leaf area duration.

Coomasie-stained 2D-polyacrylamide gel electrophoresis (PAGE).

*H. Tuberosus*, and others.

correlation (*p* < 0.01 *R*<sup>2</sup>

used were physiological parameters.

stability in sunflower yield under stress conditions.

using Pearson's correlation test (*p* < 0.04, *r* = 0.78).

The use of the stay-green criterion involves the selection of not only genotypes resistant to drought but also those resistant to *Macrophomina*, which tends to be a problem under stress conditions. Also, genotypes resistant to *Phomopsis* may be simultanously selected, as confirmed by the inbred lines Ha-48, Ha-22, CMS-1-40, PH-BC-2-91, PR-ST-3, RHA-SES, RHA-483, etc. as well as the hybrids made from these lines, which combine several resistance systems. Vrânceanu [21] confirmed the validity of using the stay-green criterion in the selection for drought resistance [22].

Petrović *et al.* [23] concluded that nitrate reductase activity and free-proline accumulation rate, which underwent large modifications in plants under water stress, may serve as parameters for the evaluation of sunflower genotypes for drought tolerance.

Working on the determination of water stress index in sunflower, Orta *et al.* [24] found statistically significant correlations between CWSI (crop water stress index) calculated from single leaf temperatures on the one hand and stomatal resistance, leaf area index, and available water in the root on the other.

Early sunflower hybrids generally have lower leaf area index, total evapotranspiration, and yield potential than the later hybrids. However, according to Škorić [11], early hybrids are typically sensitive to *Macrophomina*, so in the case of an early manifestation of drought they become infected and thus the advantage of earliness is nullified.

Some breeders believe that drought avoidance can be achieved by developing very early sunflower hybrids or by moving the sowing date (early or late sowing) in order to avoid the dry period. Dehydration avoidance can be achieved in several ways, for example, by selecting genotypes with reduced transpiration (water savers) or by increasing the uptake of available water from the soil by a powerful root system (water spenders).

Characteristics that appear to be correlated with drought tolerance include deeper rooting depth and more efficient root uptake of water, tolerance to high osmotic pressure, low transpiration rates, and plant ability to recover after wilting under heat stress.

The genetics of sunflower resistance to drought has not been studied sufficiently, despite numerous attempts and use of different plant characteristics. It appears safe to say that the drought resistance (tolerance) is controlled by a set of genes.

#### **1.3. Sunflower breeding for resistance to salinity**

Abiotic stress can be generated by mineral salts, which affect a considerable portion of the global arable land. Salinity ranks second after moisture stress. This stress may occur in the form of a specific mineral deficiency or toxicity, or as accumulation of an excess amount of soluble salts in the root zone [1].

Sunflowers are grown on low-to-medium-saline soils in many countries. These countries face soil salinity as a serious limiting factor in sunflower production. However, it should be remembered that there are several wild *Helianthus* species that naturally grow on saline soils. These species are important sources of genes for resistance to salinity. Breeders should apply effective screening methods in order to identify the wild species that possess genes useful in breeding for salinity resistance and equally effective breeding methods to transfer these genes into cultivated sunflower genotypes [22].

Seiler [25] stated that several wild species of *Helianthus* are native to salt-impacted habitats and may possess genes for salt tolerance. The same author reports that Chandler and Jan [26] evaluated three wild *Helianthus* species for salt tolerance, namely *H. paradoxus, H. Debilis,* and *H. annuus* population native to salty desert areas, and obtained the following results. *Helianthus debilis* tolerated a salt concentration about the same as cultivated sunflower, wilting at an NaCl concentration of 240–400 mM. The wild ecotype of *H. annuus* had a higher tolerance, with some plants surviving the NaCl concentration of 800 mM. *Helianthus paradoxus* was highly salt tolerant, with some plants surviving at 1300 mM of NaCl. Salt tolerance was a dominant trait in hybrids between *H. paradoxus* and cultivated *H. annuus*, which did as well as the wild parent.

The emergence percentage, emergence index, shoot length, and shoot fresh weight can be used as selection criteria for salt tolerance in sunflower at the seedling stage [27].

Tolerance of sunflower genotypes to salinity has been investigated by a number of researchers. Prakash *et al.* [28] found that turgor is not correlated with salt tolerance. The accumulation of proline shows a higher impact on tolerance to salinity. Since callus development, seed germination, and vigor are associated, the former could be a more reliable index of salt tolerance.

The involvement of turgor and proline in salt tolerance seems to be doubtful [29]. Prakash *et al.* [28] stated that turgor cannot be related to salt tolerance. However, proline accumulation seems to be more due to the effect of salinity.

Evidently, using *H. paradoxus* and possibly some other wild *Helianthus* species, sunflower breeders can successfully achieve high resistance to salinity. It is important to determine the selection criteria that can be applied in the breeding program, and these can be cell survival, seed germination, dry matter accumulation, leaf death or senescence, leaf ion content, leaf necrosis, root growth, osmoregulation, etc. [1].

#### **1.4. Sunflower breeding for resistance to mineral deficiency and mineral toxicity**

Sunflowers require only 10 macroelements (C, O, H, N, P, K, S, Ca, Fe, and Mg) and 6 micro‐ elements (B, Mn, Cu, Zn, Mo, and Co) for their growth and development. Air and water are the sources of carbon, oxygen, and hydrogen. The rest of the elements are taken up from the soil or fertilizers and are divided into primary elements, secondary elements, and microele‐ ments [14]. Sunflower nutrition has been the subject of many books and scientific papers, which have established optimum levels of each individual macro- and microelement needed for the normal growth and development of sunflower on different types of soil. There is also volu‐ minous literature on the deficiencies or excess levels (toxicity) of individual elements and how they affect sunflower growth and development.

Studying the diversity of elements in sunflower inbred lines, Sarić *et al.* [30] came to the conclusion that the genetic specificity for mineral nutrition is manifested not only through different contents of mineral elements but also through their distribution into individual plant organs.

As there are unfortunately no major breeding programs anywhere in the world that deal specifically with sunflower resistance to mineral deficiency and mineral toxicity, sunflower breeders should consider a possibility of establishing one or more such programs. They would have to choose appropriate breeding methods and targets, define selection criteria, and select potential resistance sources (most likely wild *Helianthus* species) [16].

### **1.5. Sunflower breeding for heat resistance**

remembered that there are several wild *Helianthus* species that naturally grow on saline soils. These species are important sources of genes for resistance to salinity. Breeders should apply effective screening methods in order to identify the wild species that possess genes useful in breeding for salinity resistance and equally effective breeding methods to transfer these genes

Seiler [25] stated that several wild species of *Helianthus* are native to salt-impacted habitats and may possess genes for salt tolerance. The same author reports that Chandler and Jan [26] evaluated three wild *Helianthus* species for salt tolerance, namely *H. paradoxus, H. Debilis,* and *H. annuus* population native to salty desert areas, and obtained the following results. *Helianthus debilis* tolerated a salt concentration about the same as cultivated sunflower, wilting at an NaCl concentration of 240–400 mM. The wild ecotype of *H. annuus* had a higher tolerance, with some plants surviving the NaCl concentration of 800 mM. *Helianthus paradoxus* was highly salt tolerant, with some plants surviving at 1300 mM of NaCl. Salt tolerance was a dominant trait in hybrids between *H. paradoxus* and cultivated *H. annuus*, which did as well as the wild parent.

The emergence percentage, emergence index, shoot length, and shoot fresh weight can be used

Tolerance of sunflower genotypes to salinity has been investigated by a number of researchers. Prakash *et al.* [28] found that turgor is not correlated with salt tolerance. The accumulation of proline shows a higher impact on tolerance to salinity. Since callus development, seed germination, and vigor are associated, the former could be a more reliable index of salt

The involvement of turgor and proline in salt tolerance seems to be doubtful [29]. Prakash *et al.* [28] stated that turgor cannot be related to salt tolerance. However, proline accumulation

Evidently, using *H. paradoxus* and possibly some other wild *Helianthus* species, sunflower breeders can successfully achieve high resistance to salinity. It is important to determine the selection criteria that can be applied in the breeding program, and these can be cell survival, seed germination, dry matter accumulation, leaf death or senescence, leaf ion content, leaf

Sunflowers require only 10 macroelements (C, O, H, N, P, K, S, Ca, Fe, and Mg) and 6 micro‐ elements (B, Mn, Cu, Zn, Mo, and Co) for their growth and development. Air and water are the sources of carbon, oxygen, and hydrogen. The rest of the elements are taken up from the soil or fertilizers and are divided into primary elements, secondary elements, and microele‐ ments [14]. Sunflower nutrition has been the subject of many books and scientific papers, which have established optimum levels of each individual macro- and microelement needed for the normal growth and development of sunflower on different types of soil. There is also volu‐ minous literature on the deficiencies or excess levels (toxicity) of individual elements and how

**1.4. Sunflower breeding for resistance to mineral deficiency and mineral toxicity**

as selection criteria for salt tolerance in sunflower at the seedling stage [27].

into cultivated sunflower genotypes [22].

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

seems to be more due to the effect of salinity.

necrosis, root growth, osmoregulation, etc. [1].

they affect sunflower growth and development.

tolerance.

Singh [1] made a very good definition of the heat and cold resistance, which reads: "Each plant species, more particularly genotype, has an *optimum range of temperatures* for its normal growth and development: the specific temperatures would depend not only on the genotype but also on the stage of growth and development of a given genotype. When temperature moves beyond this optimal range, it generates *temperature stress*, i.e., temperature interferes with the performance. Temperature stress may be grouped into the following three categories: (1) heat stress, (2) chilling stress and (3) freezing stress."

Sunflower is characterized by high adaptability to high temperatures. At high temperatures, sunflower intensifies the process of transpiration so that its leaves remain relatively cool. Transpiration rate can be increased only if sufficient water is supplied and this calls for a deep and well-developed root system. Therefore, the choice of genotypes with a deep and powerful root system is an important criterion in the selection for sunflower tolerance to high temper‐ atures [22].

Another important criterion is the tolerance to intensive transpiration. For the environments in which high air temperatures frequently occur at the flowering stage, breeders should select genotypes capable of producing large quantities of pollen and maintain pollen viability under such conditions. It is also important for the pistil and its stigma, or for the disk flowers on the whole, to be tolerant to high temperatures, which ensures pollination and seed formation [22].

Yet another criterion for the selection of genotypes adapted to climates with high temperatures and air and soil drought is the capacity for high seed (formation) filling rate and rapid synthesis of oil in response to stress conditions.

In order for sunflower breeders to be able to determine the right breeding methods, targets, and selection criteria and to choose their breeding materials for selection for heat resistance, they must have a detailed knowledge of how sunflower organs respond to high temperatures. Sunflower is exposed to high temperatures in arid and semiarid conditions, which have been prevalent in much of Europe in 2007. High temperatures may be accompanied by high, but also low humidity levels.

The present knowledge on sunflower heat resistance allows sunflower breeders to define their selection criteria more easily and to search for sources of heat resistance in wild *Helianthus* species.

Breeding for resistance to high temperatures should be combined with selection for drought resistance. Intensive breeding programs on sunflower heat resistance should be organized in countries where excessive temperatures are a regular occurrence. Selection for heat resistance is an integral part of many breeding programs and is often combined with breeding for increased productivity and resistance to dominant diseases and drought [16].

#### **1.6. Sunflower breeding for resistance to low temperatures (cold)**

In many environments, crop productivity is limited by low temperatures. When temperatures remain above the freezing level, that is, >0°C, it is called chilling, while freezing describes temperatures below this level, that is, <0°C.

For sunflower, it is important to increase its resistance to cold in the early stages of growth and development, that is, at germination, emergence, and the stage of two to three leaf pairs, so as to enable successful early sowing. Cold resistance at maturation should be increased as well in order to enable sunflower growing at higher altitudes and in colder regions. Sources of cold resistance should be sought exclusively in the wild *Helianthus* species that are found growing wild in the mountains where winters are harsh and springs are cold [16].

Apart from wild *Helianthus* species, induced mutations can also be successfully used as sources of resistance to low temperatures.

Excellent results in the development of sunflower genotypes resistant to cold were achieved by Kalaydzhyan *et al.* [31, 32], who applied induced mutations by chemical mutagens, first of all DMS. Resistance to low temperatures was tested in 44,000 seeds of about 2.000 mutagenic progenies by planting them in late fall/early winter. Some 499 plants from 72 mutagenic progenies (0.91%) survived the harsh winter and low temperatures (down to −20°C). The following mutants showed highest resistance to low temperatures:


In any case, Kalaydzhyan *et al.* [31, 32] evidently developed a unique germplasm, which can be used for the development of winter genotypes and genotypes tolerant to low temperatures. Unfortunately, sunflower geneticists and breeders around the world seem to be unaware of these outstanding results.
