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http://journals.tubitak.gov.tr/agriculture/

Turkish Journal of Agriculture and Forestry Turk J Agric For(2014) 38: 15-22© TÜBİTAKdoi:10.3906/tar-1210-91

Utilization of related wild species (Echinacea purpurea) for genetic enhancement of cultivated sunflower (Helianthus annuus L.)

Roumiana VASSILEVSKA-IVANOVA, Boris KRAPTCHEV*, Ira STANCHEVA, Maria GENEVA,Ivan ILIEV, Georgi GEORGIEV

Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Sofia, Bulgaria

* Correspondence: bkraptchev@yahoo.com

1. IntroductionWide hybridization, including both interspecific and intergeneric hybridization, is one of the most important strategies for creating variations in plant species since it has the potential to combine useful traits, i.e. favorable morphology, disease resistance, and some environmental tolerances of both parents that could not be achieved by crossing within a single species. Sunflower (Helianthus annuus L.) cultivars lack acceptable levels of genetic variation since hybrid breeding utilizes a comparatively narrow genetic base. Wild Helianthus species are a potential source of genes for resistance or tolerance to insects, disease, and pests; for early maturation and resistance in unfavorable environmental factors (soil salinity, acidity, and drought); and as sources of cytoplasmic male sterility and fertility restoration (Faure et al. 2002; Seiler and Gulya 2004). Successful interspecific transfer of traits from wild species to cultivated sunflower was a reason for attempts of wider crosses, including those between members of related genera.

Advances in intergeneric crosses in tomato (Rick et al. 1986), species of wild grasses from the genera Aegilops, Agropyron, and Secale (Fedak 1984); wheat; rapeseed (Brassica napus); and tobacco (Nicotiana tabacum) have been well documented (Goodman et al. 1987). There

is evidence that intentional crosses between species in different genera have the potential for crop improvement and to increase the genetic variability of crop species, including for H. annuus in particular. It is plausible that gene transfer between members of different genera may complement the more classical hybrids that provide gene flow across interspecific crosses. Additionally, they have proved to be very useful for elucidating the evolutionary relationships between H. annuus and related wild species of Asteraceae.

The present work is a part of a sunflower research program with the objective of producing and evaluating new interspecific or intergeneric hybrids that provide novel combination of traits useful for plant breeding. In particular, the program combines interest from both plant breeding and academic research in the use of wide hybridization for transferring desirable traits from wild relatives to cultivated sunflower, developing germplasm pools having wild Helianthus genes in a domestic background, and for characterizing phylogenetic affinity. Hybrids between cultivated H. annuus and species of different genera of the family Asteraceae such as Tithonia rotundifolia and Verbesina encelioides, identified as having intermediate characteristics, have been reported in our previous studies (Christov and Vassilevska-Ivanova 1999;

Abstract: Helianthus annuus is one of the most important oil species, with a comparatively narrow genetic base. The development of new sunflower cultivars is the focus of many research and breeding programs. Intergeneric hybridization between H. annuus and Echinacea purpurea, a valuable medicinal plant, has not yet been utilized in cultivar development. This paper describes 2 advanced hybrid lines produced from an intergeneric cross between the cultivated sunflower inbred line 6650 and an accession of E. purpurea. The hybrid plants have successfully been grown in the field. The hybrids, showing expression of traits from both parental species, were intermediate between both parents for plant height, leaf shape, leaf color, floral characteristics, tocopherol content, and fatty acid profile. It could be postulated that the hybridization Helianthus × Echinacea offers opportunities for combining desirable traits that increase the variability of cultivated sunflower.

Key words: Antioxidant content, cultivated sunflower, Echinacea purpurea, fatty acid, Helianthus annuus, wide hybridization

Received: 02.11.2012 Accepted: 13.05.2013 Published Online: 13.12.2013 Printed: 20.01.2014

Research Article

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Vassilevska-Ivanova et al. 1999; Vassilevska-Ivanova and Tcekova 2005; Vassilevska-Ivanova 2005; Vassilevska-Ivanova et al., 2013).

Herewith we present data on the wide hybridization between cultivated sunflower and Echinacea purpurea (L.) Moench, one of the coneflowers of the genus Echinacea. Coneflowers are a group of native American wildflowers from the daisy family (Asteraceae) characterized by spiny flowering heads with an elevated receptacle that forms the “cone”. We chose Echinacea purpurea for several reasons: 1) it has a high medicinal value worldwide, resulting from the combined effects of several phytochemicals; 2) as a model of wide hybridization between 2 distantly related genera of Asteraceae, its successful cross with sunflower would be of substantial benefit for plant science and plant breeding; 3) the intriguing aspect of wide hybridization as a powerful tool for the development of new useful genetic resources and crop improvement; and 4) modern breeding has renewed the interest for wide hybridization-derived materials with molecular assisted selection to release adaptive and high-yielding cultivars.

There are 9 species of Echinacea, all of which are perennials. Being largely used in traditional medicine, E. pallida, E. purpurea, and E. angustifolia have been the most investigated of these species. E. purpurea, or “purple” coneflower, is a clump-forming herbaceous plant with chromosome number 2n = 22. The daisy-like flower heads are very attractive with rose-purple rays and large, cone-shaped purple-brown centers (American Botanical Council, 1994; Greenfield and Davies, 2004).

This paper reports on 2 intergeneric Helianthus × Echinacea hybrid lines produced by conventional crosses. Their distinguished phenotype and agronomic traits as well as antioxidant potential and fatty acid composition indicated that these lines have the potential to provide insight into the processes and consequences attendant to wide hybridization. Strategies for utilizing members of different genera in conventional sunflower breeding programs for crop improvement are discussed.

2. Materials and methods2.1. Plant material Both intergeneric lines originated from pollination of cytoplasmic male sterile line L 6650 of cultivated sunflower Helianthus annuus (female), released by the Dobrudja Agricultural Institute, Bulgaria, with bulked pollen from wild perennial Echinacea purpurea (male). The population of E. purpurea used in this study was recovered from the genetic collection of the Institute for Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH, Zürich, Switzerland. The cross was made using the conventional hybridization method. Each experiment was carried out on flower heads that had been

protected from foreign pollen by bagging. First-generation hybrid plants were verified using morphological and cytological methods, and F1 hybrids were back-crossed to common sunflower to obtain BC1 and BC2. Some BC2 progeny possessed a new phenotype (light green dwarfed plants with specifically serrated leaves) that was not observed in the parents. These traits were selected for and fixed after BC2 to produce 2 introgression lines (branched and unbranched). Seeds from advanced plant generations were produced after self-pollination under a bag. The plant growth conditions that were employed have already been described (Vassilevska-Ivanova and Naidenova 2005). Both lines represented here are F8 progeny of selfed plants; they were raised in the experimental field of the Institute of Plant Physiology and Genetics, Sofia, Bulgaria (42°50′N, 23°0′E, altitude of 595 m) during 2010–2012. Seeds were sown in 5-m-long rows spaced 0.70 m apart with 3 replications. The sowings were performed in mid-April. Conventional management practices were used. Morphological traits were recorded for 30 random plants from both lines and their parents. The methods used in this investigation for hybridizing plants, fertility tests, and morphological comparisons are the same as those described in previous reports (Vassilevska-Ivanova and Tcekova 2005). 2.2. Phenotype observationsThirty randomly selected plants from each line and both parents were used for recording days to flower (50%), self-compatibility (%), head diameter (cm), branched or not branched, stainability of pollen using the acetocarmine test, 1000-seed weight (g), absolute dry matter (%), and seed protein content (N × 6.25) calculated by the standard method. Morphological floral characteristics included number of ray flowers, length of the corolla of ray flowers (cm), and width of the corolla of ray flowers (cm). All floral characteristics were measured at the end of anthesis. 2.3. Antioxidants analysesThe total antioxidant capacity (free radical scavenging activity) as well as the presence of ascorbate and tripeptide thiol, glutathione (α-glutamyl cysteinylglycine), tocopherols, phenols, and flavonoids was determined in leaves from 10 plants (bulked probe) of the hybrid lines and parents. All methods used were previously described (Stancheva et al. 2011). The ascorbate, glutathione, and tocopherol content were assayed spectrophotometrically using a slightly modified method based on absorption of a phosphomolybdenum complex proposed by Prieto et al. (1999). The total antioxidant capacity was measured by bleaching the purple-colored methanol solution of free stable radical (2,2-diphenyl-1-picryl-hydrazyl, DPPH) following the method of Tepe et al. (2006).

DPPH is a stable radical with a maximum absorption at 517 nm that can readily undergo reduction by an

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antioxidant. Antioxidant activity of the sample was calculated as percent inhibition of oxidation versus control sample (blank), using the following equation:

% antioxidant activity (I) = (Ablank – Asample / Ablank) × 100,

where Ablank is the absorbance of the control sample (containing all reagents except the test compound), and Asample is the absorbance of the plant extracts.

The concentration of total phenols in the extracts was measured using the Folin–Ciocalteu method and calculated as caffeic acid equivalents (Pfeffer et al. 1998). The results were expressed in milligrams of caffeic acid per gram of dry weight. The total flavonoid content in the ray flowers was measured by Zhishen et al. (1999) using a standard curve with catechin as the standard. Total flavonoid content was expressed in milligrams of catechin equivalents per gram of dry weight. 2.4. Analysis of total lipids and fatty acid composition in fresh leavesThe fresh biomass from 15 plants was extracted 3 times with chloroform-methanol (2:1 v/v) for 0.5 h under reflux. The extract was evaporated in vacuo and the residue was re-extracted with chloroform to obtain the total lipids, which were estimated gravimetrically and presented as percent of fresh biomass. The lipid samples were converted to fatty acid methyl esters by heating in methanol containing 6% anhydrous HCl at 60 °C for 1.5 h. The fatty acid methyl esters were extracted with hexane and purified by thin layer chromatography on silica gel with hexane/diethyl

ether at 10:1. The fatty acids were analyzed in triplicate using gas chromatography on a PerkinElmer instrument as previously described elsewhere (Iliev and Petkov 2006). The fatty acids were identified using reference substances. 2.5. Statistical analysisData from biochemical parameters were subjected to one-way analysis of variance for comparison of means, and significant differences were calculated according to Fisher’s least significant difference test at the 5% level using Statigraphics Plus, version 5.1. Experimental results were expressed as mean ± standard error.

3. Results 3.1. Phenotype characteristic and agronomic traitsThe low cross-compatibility level between H. annuus and Echinacea purpurea was reported in our previous paper (Vassilevska-Ivanova and Naidenova 2005). Numerous attempts at crossing different cultivated sunflower lines (HA 89, L 1234, L 1607) and populations of E. purpurea were unsuccessful since no seeds were obtained. From all possible crosses carried out, only H. annuus L 6650 × E. purpurea produced a seed set. While difficulties were evident when Echinacea was used as a female parent and crosses failed, we supposed that both parental species are cross-compatible only in one direction (Vassilevska-Ivanova and Naidenova 2005).

Developing hybrid plants showed many phenotypic features intermediate of both parents (Figure 1). This suggests that characters from both parents are being

a b

Figure 1. Plants of the 2 intergeneric lines Helianthus × Echinacea: a) unbranched, b) branched.

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expressed. The most obvious features of the hybrid phenotypes include greatly reduced internode length and stature. Apart from their heights, one of the lines was branched (multi-headed) with a well-differentiated central stem as the lateral branches arose principally on the base of the stem and petioles. The other line was single-headed without axial branches (Figure 1). The hybrid nature of the plants affected their habit, such as in internode length, shape and color of the leaves, leaf size, and lateral growth. Despite being an intermediate phenotype (Table 1), the plants appear to be of cultivated sunflower type, resembling H. annuus for some species-specific morphological traits.

The reduction in stem length exceeded 50%, resulting in a mature height of approximately 66.4 cm (branched line) and 76.0 cm (unbranched) (Table 1). The leaves of the branched line were of H. annuus type: well-developed, bright-green, but smaller than those in the sunflower. It was interesting to realize that all the leaves of the branched line were strongly serrated with small pointy teeth that point toward the tip of the leaf. This leaf shape might be

due to the complementary gene interaction between the 2 distantly related species. Moreover, the flower heads that grow apically on the branched line had a smaller diameter compared to the unbranched line, reaching only 10.5 cm. Thus, the branched line could be classified as dwarf, having internodes reduced by more than 50% of the standard length of a cultivated sunflower. The unbranched phenotype resembles the characteristic features of the semidwarf phenotypes but the other plant organs are of normal size. For example, the leaves of the unbranched line were ovate and normal green as those of cultivated sunflower and the head diameter reached 18.0 cm (Table 1). None of the parental habits were dwarf; however, self-crossed progeny of the distantly related parents revealed that probably one of the parents (H. annuus or E. purpurea) carried the dwarf phenotype in heterozygous configuration. The strongly biased plant stature towards the male parent E. purpurea may also explain, at least partly, the dwarf and semidwarf phenotypes. Dwarfism is a characteristic considered desirable in sunflower, as it gives

Table 1. Agronomic traits of H. annuus, E. purpurea, and intergeneric lines Helianthus × Echinacea.

Traits

H. annuus ×E. purpurea(branched) mean ± SD**

H. annuus ×E. purpurea (unbranched) mean ± SD

H. annuusL 6650mean ± SD

E. purpureamean ± SD

Flowering:

Days to bloom, 50% 74 76 90 72

Self-compatibility, % 90 93 0 90

Floral characteristics:

Number of ray flowers 33.4 ± 5.95e 20.3 ± 0.47a 48.0 ± 0.14a 28.0 ± 0.18a

Length of the corolla of ray flowers, cm 4.3 ± 0.22a 4.6 ± 0.61b 7.4 ± 0.05a 3.6 ± 0.33a

Width of the corolla of ray flowers, cm 1.9 ± 0.13a 3.6 ± 0.34b 3.2 ± 0.14a 1.0 ± 0.02a

Pollen stainability, % 92 94 0 90

Maturity:

Plant height, cm 66.4 ± 6.30c 76.0 ± 2.43b 153.6 ± 2.10b 85.0 ± 1.70a

Number of branches 20.5 ± 2.70c 0 0 13.0 ± 0.34a

*Head diameter, cm 10.5 ± 0.41a 18.0 ± 0.69b 24.2 ± 0.36a 4.8 ± 0.52b

Postharvest:

1000-seed weight, g 27.0 ± 0.77b 57.1 ± 1.12d 101.3 ± 0.37b 4.2 ± 0.03a

Crude protein content, % 29.4 ± 0.09a 32.3 ± 0.18b 20.67 ± 0.13b no data

Absolute dry matter, % 93.6 ± 0.20a 94.77 ± 0.21a 94.83 ± 0.25a no data

*diameter of apical head, **SD = standard deviation.

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some advantages such as higher lodging resistance and easier cultivation and harvest (Vassilevska and Tcekova 2005; Davey and Masood 2010).

Both the branched and unbranched lines flower early, supporting the positive correlation between days to flower and plant height in accordance with the results reported by Toms et al. (1992) and Toms and Pooni (1995), where they indicated that short lines flower comparatively earlier than normal height sunflower lines. Furthermore, the branched and unbranched lines both have high pollen stainability (92% and 94%, respectively).

The number of ray (ligulate) flowers of the outer whorl of the head in H. annuus is genetically determined (Luczkiewicz 1975) and can be considered to be a specific qualitative trait characterizing putative hybrids (Fambrini et al. 2003). Therefore, to determine the effect of the intergeneric cross, we evaluated the new sunflower lines by their morphological floral characteristics (Table 1). Generally, the plants produced inflorescences and flowers with normal structure (albeit somewhat reduced in size); however, differences in the number of ray flowers and width of the corolla of ray flowers became apparent between both lines and parents (H. annuus and E. purpurea), thus indicating that the change of floral characteristics in the observed lines was restricted to ray flowers. Moreover, there was a significant difference in the number and size of ligulate ray flowers between the branched and unbranched lines. As a result, the width of the corolla was markedly smaller (almost 3-fold) in the branched line than in the unbranched line. 3.2. Antioxidants analyses and fatty acid compositionThere was no significant difference in antioxidant activity as measured by the DPPH radical scavenging method between cultivated sunflower and the branched and unbranched lines (Table 2). In both lines, total ascorbic acid concentration was higher than in cultivated sunflower (Table 2). Wild plants are typically known to have higher levels of vitamin C than cultivated ones (Eaton and Konner

1985), and results of the ascorbic acid level test for leaves revealed that hybridization between cultivated sunflower and wild E. purpurea could have occurred. Increased ascorbic acid level may have resulted in greater resistance of these lines to oxidative stress than the cultivated sunflower. The total phenolic content of the hybrid lines and the 2 parental species in terms of caffeic acid equivalents is presented in Table 2. It was observed that the phenolic content was markedly higher in E. purpurea than in the cultivated sunflower H. annuus and hybrid lines, thus confirming the high antioxidant activity of E. purpurea. The flavonoid contents of the parental species and hybrid lines are given in Table 2. The unbranched line H. annuus × E. purpurea had the highest value of 2.46 mg catechin per gram of dry weight and H. annuus had the lowest value of 1.97 mg catechin per gram of dry weight. The results also indicated that while E. purpurea dry leaves contain high phenolics, only 7.84% of these compounds were flavonoids and the highest amount of phenolics were nonflavonoids. The total tocopherol content data (Table 2) obtained from the parents and 2 hybrid lines indicate that the hybridization significantly affected this trait. The difference in tocopherol content is clearly expressed in the lines and between parents, while E. purpurea has markedly low content of tocopherols.

The fatty acid profiles of the hybrid and parent fresh leaves are presented in Table 3. Variations in the contents of palmitic (C16:0) and stearic (C18:0) saturated fatty acids with regard to the parental species were observed in both the branched and unbranched lines (Table 3). The hybrids contain lauric acid at levels lower than those found in the E. purpurea parent. H. annuus does not produce this type of saturated acid, providing further evidence that characters from both parents are expressed in the hybrids. Analysis of unsaturated fatty acid composition in parents and both lines revealed a number of differences between these genotypes (Table 3).

Table 2. Antioxidant content in the leaves of H. annuus, E. purpurea, and intergeneric lines Helianthus × Echinacea.

Antioxidants H. annuus ×E. purpurea (branched)

H. annuus × E. purpurea (unbranched)

H. annuusL 6650 E. purpurea

DPPH (%) 91.57 ± 4.58a 90.42 ± 4.52a 90.63 ± 4.43a 86.59 ± 4.33a

ASC (μM g DW–1) 113.72 ± 5.69b 121.28 ± 6.06bc 93.63 ± 4.68a 168.23 ± 8.41e

Glu (μM g DW–1) 143.21 ± 7.16b 152.72 ± 7.64bc 117.90 ± 5.90a 211.73 ± 10.59e

Phenols (mg g DW–1) 18.60 ± 0.93a 18.21 ± 0.91a 18.86 ± 0.94a 29.35 ± 1.46c

Flavonoids (μM g DW–1) 2.31 ± 0.116bc 2.46 ± 0.12c 1.97 ± 0.10a 2.30 ± 0.11bc

Vitamin E (μM g DW–1) 1.92 ± 0.10b 2.79 ± 0.14c 3.69 ± 0.18d 0.50 ± 0.02a

Each value is expressed as mean ± standard deviation (n = 3).

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4. Discussion In sunflower, interspecific hybridization has successfully been used to produce new cultivars with useful traits of both parents and to incorporate desirable traits of one species into another (Seiler and Gulya 2004; Breton et al. 2012). In contrast, the use of wild gene transfer via intergeneric crosses has not been as successful so far. At the same time, for several economically important domesticated crop species, advances in intergeneric gene transfer for the production of new cultivars continue today (Hajjar and Hodgkin 2007). In this paper we have presented genetically stable lines that have intermediate characteristics between both parents and will likely be useful in sunflower breeding.

The most remarkable morphological changes observed in the Helianthus × Echinacea lines presented here were the large reduction in plant stature, the serrated, light-green leaves, and a floral morphology suggesting the pleiotropic effect of hybridization. In fact, the entire plant phenotype was changed although the species-specific characteristics of the cultivated parent H. annuus prevailed. We also identified modified traits related to antioxidant activity and fatty acid composition in leaf samples as they were altered by the intergeneric hybridization to a different extent. Some of the biochemical characteristics like ascorbic acid, glutathione, and tocopherol content were substantially affected in both hybrid lines compared with H. annuus and E. purpurea (Table 2). The most dramatic antioxidant change observed in the hybrid lines was the large augmentation in tocopherol content (up to 5-fold in unbranched plants in comparison with the male parent E. purpurea). Changes in the levels of DPPH, the phenolics, and the other monitored compounds were either small or absent (Table 2). Furthermore, neither antioxidant parameter displayed good linear

correlation in respect to total antioxidant activity. It is known that total antioxidant activity is determined by the levels of antioxidant metabolites and enzymes from the ascorbate–glutathione cycle as well as catalase, superoxide dismutase, and other peroxidases. The lack of relationship between free radical scavenging activity and low molecular antioxidants could be due to the more significant role of antioxidant enzymes in defense. These antioxidant systems can be divided into 2 categories: ones that react with reactive oxygen species and keep them at low levels (peroxidases, superoxide dismutase, and catalase), and ones that regenerate the oxidized antioxidants (ascorbate peroxidase and glutathione reductase) (Smirnoff 1993). On the other hand, increases in antioxidant enzyme activity could indicate increased oxidative stress, but they give no indication of changes in overall flux through the ascorbate–glutathione cycle. The research of fatty acid composition in both lines in comparison with cultivated sunflower and E. purpurea also indicated changes that may cause or at least substantially contribute to the intergeneric hybridization. We have several conclusions regarding the intergeneric Helianthus × Echinacea phenotype. The strongest effects in the hybrid lines were on stem elongation, branching and leaf growth (shape, size, and color), tocopherol content, and variability of fatty acid composition. The current plant materials have implications for breeding purpose and may allow the exploitation of genetic diversity of more distant species, when interspecific crosses have apparently failed. The knowledge obtained in this paper constitutes a basis for further studies in which intergeneric hybridization may also be important for evolutionary studies and consideration of phylogenetic relationships not only in Helianthus but also in other genera.

Table 3. Fatty acids profile of fresh biomass from 2 intergeneric hybrids (H. annuus × E. purpurea) and the original parents (%, m m–1).

Average fatty acid composition

H. annuus ×E. purpurea (branched)

H. annuus ×E. purpurea (unbranched)

H. annuusL 6650 E. purpurea

C12:0 0.2 0.2 0 0.4

C14:0 1.6 1.8 1.3 2.7

C16:0 17.9 15.4 14.6 12.4

C16:1 2.2 2.6 2.8 1.9

C18:0 1.9 0.2 0.5 0.5

C18:1 14.7 11.5 9.5 13.6

C18:2 59.4 74.3 67.2 67.2

Lauric acid = C12:0, myristic acid = C14:0, palmitic acid = C16:0, palmitoleic acid = C16:1, stearic acid = C18:0, oleic acid = C18:1, and linoleic acid = C18:2.

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