Turkish Journal of Agriculture and Forestry Turk J Agric For (2014) 38: 15-22 http://journals.tubitak.gov.tr/agriculture/ © TÜBİTAK Research Article doi:10.3906/tar-1210-91

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

Roumiana VASSILEVSKA-IVANOVA, Boris KRAPTCHEV*, Ira STANCHEVA, Maria GENEVA, Ivan ILIEV, Georgi GEORGIEV Institute of Physiology and Genetics, Bulgarian Academy of Sciences, Sofia, Bulgaria

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

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 betweenH. 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 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

1. Introduction is evidence that intentional crosses between species in Wide hybridization, including both interspecific and different genera have the potential for crop improvement intergeneric hybridization, is one of the most important and to increase the genetic variability of crop species, strategies for creating variations in plant species since it including for H. annuus in particular. It is plausible that has the potential to combine useful traits, i.e. favorable gene transfer between members of different genera may morphology, disease resistance, and some environmental complement the more classical hybrids that provide gene tolerances of both parents that could not be achieved by flow across interspecific crosses. Additionally, they have crossing within a single species. Sunflower (Helianthus proved to be very useful for elucidating the evolutionary annuus L.) cultivars lack acceptable levels of genetic relationships between H. annuus and related wild species variation since hybrid breeding utilizes a comparatively of . narrow genetic base. Wild Helianthus species are a potential The present work is a part of a sunflower research source of genes for resistance or tolerance to insects, program with the objective of producing and evaluating disease, and pests; for early maturation and resistance in new interspecific or intergeneric hybrids that provide unfavorable environmental factors (soil salinity, acidity, novel combination of traits useful for plant breeding. and drought); and as sources of cytoplasmic male sterility In particular, the program combines interest from both and fertility restoration (Faure et al. 2002; Seiler and Gulya plant breeding and academic research in the use of wide 2004). Successful interspecific transfer of traits from wild hybridization for transferring desirable traits from wild species to cultivated sunflower was a reason for attempts relatives to cultivated sunflower, developing germplasm of wider crosses, including those between members of pools having wild Helianthus genes in a domestic related genera. background, and for characterizing phylogenetic affinity. Advances in intergeneric crosses in tomato (Rick et al. Hybrids between cultivated H. annuus and species of 1986), species of wild grasses from the genera Aegilops, different genera of the family Asteraceae such as Agropyron, and Secale (Fedak 1984); wheat; rapeseed rotundifolia and Verbesina encelioides, identified as having (Brassica napus); and tobacco (Nicotiana tabacum) have intermediate characteristics, have been reported in our been well documented (Goodman et al. 1987). There previous studies (Christov and Vassilevska-Ivanova 1999; * Correspondence: [email protected] 15 VASSILEVSKA-IVANOVA et al. / Turk J Agric For

Vassilevska-Ivanova et al. 1999; Vassilevska-Ivanova and protected from foreign pollen by bagging. First-generation Tcekova 2005; Vassilevska-Ivanova 2005; Vassilevska- hybrid plants were verified using morphological and

Ivanova et al., 2013). cytological methods, and F1 hybrids were back-crossed

Herewith we present data on the wide hybridization to common sunflower to obtain BC1 and BC2. Some BC2 between cultivated sunflower and Echinacea purpurea (L.) progeny possessed a new phenotype (light green dwarfed Moench, one of the coneflowers of the genus Echinacea. plants with specifically serrated leaves) that was not Coneflowers are a group of native American wildflowers observed in the parents. These traits were selected for and from the daisy family (Asteraceae) characterized by spiny fixed after 2BC to produce 2 introgression lines (branched flowering heads with an elevated receptacle that forms the and unbranched). Seeds from advanced plant generations “cone”. We chose Echinacea purpurea for several reasons: were produced after self-pollination under a bag. The 1) it has a high medicinal value worldwide, resulting from plant growth conditions that were employed have already the combined effects of several phytochemicals; 2) as a been described (Vassilevska-Ivanova and Naidenova model of wide hybridization between 2 distantly related 2005). Both lines represented here are F8 progeny of selfed genera of Asteraceae, its successful cross with sunflower plants; they were raised in the experimental field of the would be of substantial benefit for plant science and plant Institute of Plant Physiology and Genetics, Sofia, Bulgaria breeding; 3) the intriguing aspect of wide hybridization as (42°50′N, 23°0′E, altitude of 595 m) during 2010–2012. a powerful tool for the development of new useful genetic Seeds were sown in 5-m-long rows spaced 0.70 m apart resources and crop improvement; and 4) modern breeding with 3 replications. The sowings were performed in mid- has renewed the interest for wide hybridization-derived April. Conventional management practices were used. materials with molecular assisted selection to release Morphological traits were recorded for 30 random plants adaptive and high-yielding cultivars. from both lines and their parents. The methods used in There are 9 species of Echinacea, all of which are this investigation for hybridizing plants, fertility tests, perennials. Being largely used in traditional medicine, and morphological comparisons are the same as those E. pallida, E. purpurea, and E. angustifolia have been described in previous reports (Vassilevska-Ivanova and the most investigated of these species. E. purpurea, or Tcekova 2005). “purple” coneflower, is a clump-forming 2.2. Phenotype observations with chromosome number 2n = 22. The daisy-like flower Thirty randomly selected plants from each line and both heads are very attractive with rose-purple rays and large, parents were used for recording days to flower (50%), self- cone-shaped purple-brown centers (American Botanical compatibility (%), head diameter (cm), branched or not Council, 1994; Greenfield and Davies, 2004). branched, stainability of pollen using the acetocarmine This paper reports on 2 intergeneric Helianthus × test, 1000-seed weight (g), absolute dry matter (%), and Echinacea hybrid lines produced by conventional crosses. seed protein content (N × 6.25) calculated by the standard Their distinguished phenotype and agronomic traits as method. Morphological floral characteristics included well as antioxidant potential and fatty acid composition number of ray flowers, length of the corolla of ray flowers indicated that these lines have the potential to provide (cm), and width of the corolla of ray flowers (cm). All insight into the processes and consequences attendant floral characteristics were measured at the end of anthesis. to wide hybridization. Strategies for utilizing members 2.3. Antioxidants analyses of different genera in conventional sunflower breeding The total antioxidant capacity (free radical scavenging programs for crop improvement are discussed. activity) as well as the presence of ascorbate and tripeptide thiol, glutathione (α-glutamyl cysteinylglycine), 2. Materials and methods tocopherols, phenols, and flavonoids was determined in 2.1. Plant material leaves from 10 plants (bulked probe) of the hybrid lines Both intergeneric lines originated from pollination and parents. All methods used were previously described of cytoplasmic male sterile line L 6650 of cultivated (Stancheva et al. 2011). The ascorbate, glutathione, and sunflower Helianthus annuus (female), released by the tocopherol content were assayed spectrophotometrically Dobrudja Agricultural Institute, Bulgaria, with bulked using a slightly modified method based on absorption of pollen from wild perennial Echinacea purpurea (male). a phosphomolybdenum complex proposed by Prieto et al. The population of E. purpurea used in this study was (1999). The total antioxidant capacity was measured by recovered from the genetic collection of the Institute bleaching the purple-colored methanol solution of free for Pharmaceutical Sciences, Swiss Federal Institute of stable radical (2,2-diphenyl-1-picryl-hydrazyl, DPPH) Technology, ETH, Zürich, Switzerland. The cross was following the method of Tepe et al. (2006). made using the conventional hybridization method. Each DPPH is a stable radical with a maximum absorption experiment was carried out on flower heads that had been at 517 nm that can readily undergo reduction by an

16 VASSILEVSKA-IVANOVA et al. / Turk J Agric For antioxidant. Antioxidant activity of the sample was ether at 10:1. The fatty acids were analyzed in triplicate calculated as percent inhibition of oxidation versus control using gas chromatography on a PerkinElmer instrument sample (blank), using the following equation: as previously described elsewhere (Iliev and Petkov 2006). The fatty acids were identified using reference substances. % antioxidant activity (I) = (Ablank – Asample / Ablank) × 100, 2.5. Statistical analysis where A is the absorbance of the control sample blank Data from biochemical parameters were subjected to (containing all reagents except the test compound), and one-way analysis of variance for comparison of means, Asample is the absorbance of the plant extracts. and significant differences were calculated according to The concentration of total phenols in the extracts Fisher’s least significant difference test at the 5% level using was measured using the Folin–Ciocalteu method and Statigraphics Plus, version 5.1. Experimental results were calculated as caffeic acid equivalents (Pfeffer et al. 1998). expressed as mean ± standard error. The results were expressed in milligrams of caffeic acid per gram of dry weight. The total flavonoid content in the 3. Results ray flowers was measured by Zhishen et al. (1999) using 3.1. Phenotype characteristic and agronomic traits a standard curve with catechin as the standard. Total The low cross-compatibility level betweenH. annuus and flavonoid content was expressed in milligrams of catechin Echinacea purpurea was reported in our previous paper equivalents per gram of dry weight. (Vassilevska-Ivanova and Naidenova 2005). Numerous 2.4. Analysis of total lipids and fatty acid composition in attempts at crossing different cultivated sunflower lines fresh leaves (HA 89, L 1234, L 1607) and populations of E. purpurea The fresh biomass from 15 plants was extracted 3 times were unsuccessful since no seeds were obtained. From with chloroform-methanol (2:1 v/v) for 0.5 h under reflux. all possible crosses carried out, only H. annuus L 6650 × The extract was evaporated in vacuo and the residue was re- E. purpurea produced a seed set. While difficulties were extracted with chloroform to obtain the total lipids, which evident when Echinacea was used as a female parent and were estimated gravimetrically and presented as percent crosses failed, we supposed that both parental species of fresh biomass. The lipid samples were converted to fatty are cross-compatible only in one direction (Vassilevska- acid methyl esters by heating in methanol containing 6% Ivanova and Naidenova 2005). anhydrous HCl at 60 °C for 1.5 h. The fatty acid methyl Developing hybrid plants showed many phenotypic esters were extracted with hexane and purified by thin features intermediate of both parents (Figure 1). This layer chromatography on silica gel with hexane/diethyl suggests that characters from both parents are being

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

17 VASSILEVSKA-IVANOVA et al. / Turk J Agric For expressed. The most obvious features of the hybrid due to the complementary gene interaction between the phenotypes include greatly reduced internode length and 2 distantly related species. Moreover, the flower heads stature. Apart from their heights, one of the lines was that grow apically on the branched line had a smaller branched (multi-headed) with a well-differentiated central diameter compared to the unbranched line, reaching only stem as the lateral branches arose principally on the base 10.5 cm. Thus, the branched line could be classified as of the stem and petioles. The other line was single-headed dwarf, having internodes reduced by more than 50% of the without axial branches (Figure 1). The hybrid nature of standard length of a cultivated sunflower. The unbranched the plants affected their habit, such as in internode length, phenotype resembles the characteristic features of the shape and color of the leaves, leaf size, and lateral growth. semidwarf phenotypes but the other plant organs are of Despite being an intermediate phenotype (Table 1), the normal size. For example, the leaves of the unbranched plants appear to be of cultivated sunflower type, resembling line were ovate and normal green as those of cultivated H. annuus for some species-specific morphological traits. sunflower and the head diameter reached 18.0 cm (Table The reduction in stem length exceeded 50%, resulting 1). None of the parental habits were dwarf; however, in a mature height of approximately 66.4 cm (branched self-crossed progeny of the distantly related parents line) and 76.0 cm (unbranched) (Table 1). The leaves of revealed that probably one of the parents (H. annuus or the branched line were of H. annuus type: well-developed, E. purpurea) carried the dwarf phenotype in heterozygous bright-green, but smaller than those in the sunflower. It configuration. The strongly biased plant stature towards was interesting to realize that all the leaves of the branched the male parent E. purpurea may also explain, at least line were strongly serrated with small pointy teeth that partly, the dwarf and semidwarf phenotypes. Dwarfism is a point toward the tip of the leaf. This leaf shape might be characteristic considered desirable in sunflower, as it gives

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

H. annuus × H. annuus × H. annuus E. purpurea E. purpurea E. purpurea Traits L 6650 (branched) (unbranched) mean ± SD mean ± SD mean ± SD** mean ± 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.

18 VASSILEVSKA-IVANOVA et al. / Turk J Agric For some advantages such as higher lodging resistance and 1985), and results of the ascorbic acid level test for leaves easier cultivation and harvest (Vassilevska and Tcekova revealed that hybridization between cultivated sunflower 2005; Davey and Masood 2010). and wild E. purpurea could have occurred. Increased Both the branched and unbranched lines flower early, ascorbic acid level may have resulted in greater resistance supporting the positive correlation between days to flower of these lines to oxidative stress than the cultivated and plant height in accordance with the results reported by sunflower. The total phenolic content of the hybrid Toms et al. (1992) and Toms and Pooni (1995), where they lines and the 2 parental species in terms of caffeic acid indicated that short lines flower comparatively earlier than equivalents is presented in Table 2. It was observed that normal height sunflower lines. Furthermore, the branched the phenolic content was markedly higher in E. purpurea and unbranched lines both have high pollen stainability than in the cultivated sunflower H. annuus and hybrid (92% and 94%, respectively). lines, thus confirming the high antioxidant activity of E. The number of ray (ligulate) flowers of the outer whorl of purpurea. The flavonoid contents of the parental species the head in H. annuus is genetically determined (Luczkiewicz and hybrid lines are given in Table 2. The unbranched line 1975) and can be considered to be a specific qualitative H. annuus × E. purpurea had the highest value of 2.46 mg trait characterizing putative hybrids (Fambrini et al. 2003). catechin per gram of dry weight and H. annuus had the Therefore, to determine the effect of the intergeneric cross, lowest value of 1.97 mg catechin per gram of dry weight. we evaluated the new sunflower lines by their morphological The results also indicated that whileE. purpurea dry leaves floral characteristics (Table 1). Generally, the plants contain high phenolics, only 7.84% of these compounds produced inflorescences and flowers with normal structure were flavonoids and the highest amount of phenolics were (albeit somewhat reduced in size); however, differences in nonflavonoids. The total tocopherol content data (Table the number of ray flowers and width of the corolla of ray 2) obtained from the parents and 2 hybrid lines indicate flowers became apparent between both lines and parents (H. that the hybridization significantly affected this trait. The annuus and E. purpurea), thus indicating that the change difference in tocopherol content is clearly expressed in the of floral characteristics in the observed lines was restricted lines and between parents, while E. purpurea has markedly to ray flowers. Moreover, there was a significant difference low content of tocopherols. in the number and size of ligulate ray flowers between the The fatty acid profiles of the hybrid and parent fresh branched and unbranched lines. As a result, the width of the leaves are presented in Table 3. Variations in the contents corolla was markedly smaller (almost 3-fold) in the branched of palmitic (C16:0) and stearic (C18:0) saturated fatty line than in the unbranched line. acids with regard to the parental species were observed 3.2. Antioxidants analyses and fatty acid composition in both the branched and unbranched lines (Table 3). The There was no significant difference in antioxidant activity hybrids contain lauric acid at levels lower than those found as measured by the DPPH radical scavenging method in the E. purpurea parent. H. annuus does not produce this between cultivated sunflower and the branched and type of saturated acid, providing further evidence that unbranched lines (Table 2). In both lines, total ascorbic characters from both parents are expressed in the hybrids. acid concentration was higher than in cultivated sunflower Analysis of unsaturated fatty acid composition in parents (Table 2). Wild plants are typically known to have higher and both lines revealed a number of differences between levels of vitamin C than cultivated ones (Eaton and Konner these genotypes (Table 3).

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

H. annuus × H. annuus × E. purpurea H. annuus Antioxidants E. purpurea E. purpurea (branched) (unbranched) L 6650 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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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 H. annuus × H. annuus × H. annuus E. purpurea composition E. purpurea (branched) E. purpurea (unbranched) L 6650 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.

4. Discussion correlation in respect to total antioxidant activity. It is In sunflower, interspecific hybridization has successfully known that total antioxidant activity is determined by the been used to produce new cultivars with useful traits of levels of antioxidant metabolites and enzymes from the both parents and to incorporate desirable traits of one ascorbate–glutathione cycle as well as catalase, superoxide species into another (Seiler and Gulya 2004; Breton et dismutase, and other peroxidases. The lack of relationship al. 2012). In contrast, the use of wild gene transfer via between free radical scavenging activity and low molecular intergeneric crosses has not been as successful so far. antioxidants could be due to the more significant role At the same time, for several economically important of antioxidant enzymes in defense. These antioxidant domesticated crop species, advances in intergeneric gene systems can be divided into 2 categories: ones that react transfer for the production of new cultivars continue with reactive oxygen species and keep them at low levels today (Hajjar and Hodgkin 2007). In this paper we have (peroxidases, superoxide dismutase, and catalase), and presented genetically stable lines that have intermediate ones that regenerate the oxidized antioxidants (ascorbate characteristics between both parents and will likely be peroxidase and glutathione reductase) (Smirnoff 1993). On useful in sunflower breeding. the other hand, increases in antioxidant enzyme activity The most remarkable morphological changes observed could indicate increased oxidative stress, but they give no in the Helianthus × Echinacea lines presented here were the indication of changes in overall flux through the ascorbate– large reduction in plant stature, the serrated, light-green glutathione cycle. The research of fatty acid composition in leaves, and a floral morphology suggesting the pleiotropic both lines in comparison with cultivated sunflower and E. effect of hybridization. In fact, the entire plant phenotype purpurea also indicated changes that may cause or at least was changed although the species-specific characteristics of substantially contribute to the intergeneric hybridization. the cultivated parent H. annuus prevailed. We also identified modified traits related to antioxidant activity and fatty acid We have several conclusions regarding the intergeneric composition in leaf samples as they were altered by the Helianthus × Echinacea phenotype. The strongest effects in intergeneric hybridization to a different extent. Some of the the hybrid lines were on stem elongation, branching and biochemical characteristics like ascorbic acid, glutathione, leaf growth (shape, size, and color), tocopherol content, and tocopherol content were substantially affected in both and variability of fatty acid composition. The current plant hybrid lines compared with H. annuus and E. purpurea materials have implications for breeding purpose and may (Table 2). The most dramatic antioxidant change observed allow the exploitation of genetic diversity of more distant in the hybrid lines was the large augmentation in tocopherol species, when interspecific crosses have apparently failed. content (up to 5-fold in unbranched plants in comparison The knowledge obtained in this paper constitutes a basis for with the male parent E. purpurea). Changes in the levels of further studies in which intergeneric hybridization may also DPPH, the phenolics, and the other monitored compounds be important for evolutionary studies and consideration of were either small or absent (Table 2). Furthermore, phylogenetic relationships not only in Helianthus but also neither antioxidant parameter displayed good linear in other genera.

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