Cent. Eur. J. Biol. • 8(5) • 2013 • 492-498 DOI: 10.2478/s11535-013-0147-8

Central European Journal of Biology

A compact sunflower line produced after cross annuus x

Research Article

Roumiana D. Vassilevska-Ivanova*, Boris V. Kraptchev, Ira Stancheva, Maria Geneva

Institute of Physiology and Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

Received 17 October 2012; Accepted 22 January 2013

Abstract: Intergeneric cross was made between the cultivated sunflower inbred line HA89 and an accession of wild Verbesina

encelioides tolerant to drought and high temperature. The line was a BC2F5 progeny. The most remarkable feature of the  was their compact architecture due to short petiole length and also, rather specific bright-yellow inflorescences. Similar plant architecture did not exist in either the wild or the cultivated parent. For sunflower, it is considered as a favourable and potentially useful adaptive trait. The line was multi-branched of medium type branching and possessed good agronomic characteristics.TheoverallcharacteristicsofHA-VERBENClinemakeitausefulplantmaterialforresearchonwidehybridization.

Keywords: Antioxidants • Crown beard • Cultivated sunflower • Intergeneric cross • Introgressive hybridization • Wide hybridization ©VersitaSp.zo.o.

1. Introduction high temperature. It is an annual , propagating by seeds, which are produced in abundance. Its seeds Wide hybridization is frequently used as a tool to improve exhibit remarkable endurance to climatic extremes valuable agronomic traits of cultivated sunflower and survive under extremely high temperatures Helianthus annuus [1-5]. The range of wild Helianthus (38-46ºC) and soil drought during which they lie dormant available to use is extensive while the comprises in soil desiccated to below 5% moisture content [12]. 51 species, 14 annual and 37 perennial, which are V. encelioides is differentiated from the cultivated sunflower diploids, tetraploids and hexaploids [6]. The majority of by the opposite leaves on the lower part of the plant, as wide hybridization studies have examined interspecific well as smaller flower heads [13]. Besides its ornamental hybridization between closely related Helianthus value, some studies pointed out the medicinal and species, while limited information is available on economical importance of various fractions of Verbesina intergeneric hybridization and the barriers that prevent encelioides, which demonstrate considerable antibacterial, hybrid production [7-11]. antifungal, antiviral, antitumor, hypoglycemic and anti- The present work is a portion of a sunflower research implantation activities [14]. Intensive pharmacognostic program. Its objective is production and evaluation of and pharmacological investigations are in a progress new interspecific and intergeneric hybrids for transferring to ascertain therapeutic properties and medicinal use of desirable traits from wild relatives to cultivated sunflower V. encelioides roots for diabetes [15]. lines, developing germplasm pools having wild Helianthus In the course of our study on wide hybridization genes in domestic background, and evaluating sunflower Helianthus x Verbesina, we obtained a suite of diverse evolution and interspecific/intergeneric relationships. recombinants which reveal intermediate morphology and Verbesina encelioides [(Cav.) Bentham & Hooker fil. phenotype [9], or even novel features such as tubular ray ex Gray] or golden crown beard was chosen due to flowers [16]. Indeed, intergeneric hybridization involving its drought tolerance, early flowering and resistance to Verbesina ssp. is a genuine source of new sunflower hybrid

* E-mail: [email protected] 492 R.D. Vassilevska-Ivanova et al.

genotypes. Unfortunately, despite extensive classical and morphological comparisons are the same as studies of morphological development of these new lines, those described in a previous report [18]. The following little is known about the molecular genetics of this type of phenotype traits were recorded: plant height (cm), head wide hybridization hence, further confirmation is necessary diameter (cm), days to flower (50%), days to maturity to elucidate the hybrid nature of the developed lines. (50%), pollen staining using the acetocarmine test, The purpose of the study reported here was to thousand seeds weight (TSW) (g), number of branches, describe a compact (erect) sunflower line developed and morphological floral characteristics: number of by wide hybridization between Helianthus annuus and ray flowers; length of the corolla of ray flowers (cm) Verbesina encelioides. Generally, compact sunflowers and width of the corolla of ray flowers (cm). All floral have some valuable advantages, which is, they allow characteristics were measured at the end of anthesis. higher plant densities, facilitate cultivation and thus, it could increase the yield. Therefore, the knowledge of 2.3 Antioxidant analyses the such plant architecture in sunflower was essential in The total antioxidant capacity (free radicals scavenging breeding procedures. activity) as well as the presence of the antioxidants ascorbate, tocopherols, phenols and flavonoids was measured in dry leaves from both parental species H. 2. Experimental Procedures annuus and V. encelioides and hybrid line. All methods used were previously described [19]. The ascorbate and 2.1 Plant material tocopherol content were assayed spectrophotometrically The intergeneric line Helianthus x Verbesina originated by the slightly modified method based on absorption of from pollination of cytoplasmic male sterile (cms) line phosphomolibdenum complex proposed by Prieto et al. HA89 (female parent), a public oilseed sunflower [20]. Plant dry material (0.5 g) was ground with a mortar inbred maintainer line released by US Department of and pestle to a fine powder. Next, 3 mL ofdH2O was Agriculture–Agricultural Research Service, with bulked added and the suspension was homogenized, transferred pollen from wild annual Verbesina encelioides (male to tubes, and shaken for 1 h at room temperature for 1 h parent). The population of V. encelioides was grown from in the dark. The suspension was filtered and extraction seeds originally obtained from the Botanical Garden of was repeated with 3 mL of dH2O. The pellet was washed

Lisbon, Portugal. The cross was made by conventional again with 2 mL of dH2O. For lipid-soluble antioxidant hybridization method. Each experiment was carried out capacity (expressed as α-tocopherol), the procedure was on flower heads which had been protected from foreign the same but the extraction was carried out with hexane pollen by bagging. First generation hybrid plants were as a solvent. The method has been optimized and verified using morphological and cytological methods, characterized with respect to linearity interval, repetitively and F1 hybrids were back-crossed to the common and reproducibility, and molar absorption coefficients sunflower to obtain BC1 and BC2. Some BC2 progeny for the quantitation of water-soluble and lipid-soluble possessed new phenotype (compact architecture), antioxidant capacities, expressed as equivalents of which was not observed in the parents. These traits ascorbate and α-tocopherol [20]. Absorption coefficients –1 –1 were selected for and fixed after the BC2 to produce were (3.4±0.1)×103 M cm for ascorbic acid and an introgression line (namely HA-VERBENC). Seeds (4.0±0.1)×103 M–1 cm–1 for α-tocopherol. from advanced plant generations were produced after The total antioxidant capacity was measured from self-pollination under a bag. Plant growth conditions bleaching of the purple coloured methanol solution employed have already been described [17]. The line of free stable radical (2,2-diphenyl-1-picryl-hydrasyl, • represented here is an F5 progeny of selfed plants; it was DPPH) by the method of Tepe et al. [21]. DPPH is a raised in the Experimental Field of the Institute of Plant stable radical with a maximum absorption at 517 nm Physiology and Genetics, Sofia, Bulgaria (42º50´N, that can readily undergo reduction by an antioxidant. 23º00´E, 595 m above the see level) during 2010-2012. Antioxidant activity of the sample was calculated as Seeds were sown in 5 m rows spaced 0.70 m apart with percentage inhibition of oxidation versus control sample three replications. The sowings were performed in mid- (blank), using the equation

April. Conventional management practices were used. % antioxidant activity (I)=(Ablank-Asampe/Ablank)×100,

where Ablank is the absorbance of the control sample 2.2 Phenotype observations (containing all reagents except the test compound), and

The morphological traits were recorded of thirty random Asample is the absorbance of plant extracts. plants for hybrid line and its parents. Methods used in For determination of the phenols and flavonoids, dry this investigation for hybridizing plants, fertility tests leaf samples (1 g) were ground and exhaustively extracted

493 A compact sunflower line produced after cross Helianthus annuus x Verbesina encelioides

with 96% (v/v) methanol. Contents of phenolic compounds report [24]. Numerous attempts for crossing different were determined spectrophotometrically using Folin- cultivated sunflower lines (L1607, L3004, HA 341) and Ciocalteu reagent and calculated as caffeic acid equivalents populations of V. encelioides were unsuccessful since [22]. Flavonoids in plant tissues were measured by the no seeds were obtained. Crosses between the line method of Zhishen et al. [23] spectrophotometrically using HA89 and V. encelioides gave hybrid seeds only when the standard curve of catechin. the cultivated sunflower was the female parent. While difficulties were evident when Verbesina was used as 2.4 Statistical analysis a female parent and crosses failed we supposed that Data from biochemical parameters were subjected to both parental species are cross-compatible only in one one-way ANOVA analysis of variance for comparison direction [24]. of means, and significant differences were calculated The morphological characteristics and agronomic according to Fisher LSD test at P=0.05 using a performance of both parental species and the intergeneric statistical software package (StatGraphics Plus, version line are given in Table 1. Generally, cultivated H. annuus 5.1 for Windows). Experimental results were expressed and V. encelioides differ dramatically in phenotype and as means ± standard error. they are easily distinguished by numerous morphological features. Therefore, the successful cross between two distantly related species may produce distinguished new 3. Results phenotypes. In our case, plants were easily identified since they displayed intermediary traits between both 3.1 Phenotype characteristics and agronomic parental species. Nevertheless, the HA-VERBENC line traits appears to be of cultivated sunflower type, resembling The low cross-compatibility level between H. annuus and H. annuus for some species-specific morphological Verbesina encelioides has been reported in our previous traits. The most obvious feature of the line included

Line HA-VERBENC H. annuus HA V. encelioides Characteristics x ± SE ♀ ♂

Flowering:

Days to bloom (50%) 92 90 90

Self-compatibility, % (80-90)**85 0 no data

Pollen staining, % 86.5 0 91

Plant height at flowering, cm 116.5 ± 1.03a 153.6 ± 0.83 91.5 ± 0.68

Number of branches 19.57 ± 0.5 0 12-17

Floral morphology:

Number of ray flowers 48.4 ± 2.83 48.0 ± 0.93 24.0 ± 0.62

lim (42-59) (43-52) (19-27)

Length of the corolla of ray flowers, mm 42.1 ± 0.07 74.0 ± 0.02 16.6 ± 1.61

lim (30-50) (70-79) (15-17)

Width of the corolla of ray flowers, mm 15.0 ± 0.04 32.0 ± 0.87 55.0 ± 0.03

lim (10-20) (30-36) (5-7)

Maturity:

Head diameter*, cm 8.6 ± 1.07 24.2 ± 0.43 2.3 ± 0.05

Days to maturity (50%) 120 130 150

Post harvest:

1000 seeds weight, g 17.2 83.6 2.2

Table 1. Agronomic traits of an intergeneric sunflower line HA-VERBENC with inflorescences resembling to Gerbera HA.

aThe data are means ± SE; *diameter of apical head; **range (mean) measured by estimating seed set under bags.

494 R.D. Vassilevska-Ivanova et al.

compact plant architecture (width is reduced due to plant from the sunflower family (). The short petioles in comparison to a classic sunflower), and hybrid plants revealed an extended flowering time erected leaves. (axially flowers blooms well about 20 days past apical It was focused on that trait because of the head), thus facilitating pollination in hybridization plots assumption that compact and erect plant architecture providing viable pollen for an extended period of time. has been considered to be an important trait in Also, branched habits provide opportunity to cut sunflower breeding with potential to permit higher flowers over the longest possible period of time (in plantation density. Thus, it is highly believable that case of decorative cut sunflower growth). The leaves such a trait may represent a key step to ensure a were triangular-shape, dark-green, non-serrated spectacular increase in sunflower production. All the and shiny hairless. Also, they were opposite on the plants were completely branched as the branching lower part of the plants, a trait that characterized was of medium type, with a dominated central head. V. encelioides morphology. The diameter of apical head Wild parent V. encelioides presented branches on the and thousand seed weight (TSW) were also recorded. whole plant, without a main head. Generally, floral Head size of the line averaged 8.6 cm. As expected, parts of HA-VERBENC line are much smaller than that TSW was comparatively low and it amounted to of H. annuus (Table 1). They have bright yellow ray 17.2 g (Table 1). (ligulate) flowers as the center disk flower is also yellow (Figure 1). The petals are at right angle (90 degrees) 3.2 Antioxidant analyses to the disk of the flower. This flower head looks like There was no substantial difference in the level a yellow gerbera, which is a widely used decorative of antioxidant activity measured by DPPH radical

a b

c d

Figure 1. Helianthus annuus ♀ (a), V. encelioides ♂ (b), H. annuus x V. encelioides - whole plant (c), and flower head (d) in the field.

495 A compact sunflower line produced after cross Helianthus annuus x Verbesina encelioides

scavenging method between cultivated sunflower and Intermediacy of morphological characters in hybrids the hybrid line (Table 2). is concordant with an oligogenic or multigenic control Hybrid line HA-VERBENC accumulated more hypothesis of morphological trait expression in hybrids ascorbic acid than V. encelioides but the quantity of the [27]. According to Rieseberg and Ellstrand [28], only ascorbic acid was still lower than in H. annuus. The total 45% of the morphological characters are intermediate, phenolic content of hybrid line and two parental species while 45% have parental dominance and 10% are in caffeic acids equivalents is presented in Table 2. transgressive. In plants, there have been several well- The level of phenols was much higher in the hybrid line documented reports that morphological markers can HA-VERBENC as compared with both parental species, be problematic for the identification of hybrids because thus ascribing the high antioxidant activity of the hybrid of their environmental plasticity and unknown mode of line. Similarly, the level of flavonoids was again markedly expression [29,30]. Consequently, it was important to higher in HA-VERBENC compared with the parental ascertain if morphology in sunflowers is a good predictor species (Table 2). of the status of hybrids and if it constituted a valid form of Several comprehensive works reported a good independent evidence for the identification hybrid plants. correlation between phenolic compounds and total The intergeneric line HA-VERBENC has several notable antioxidants [25,26]. The total tocopherol content was features. Plants have compact and erect architecture, reduced 2-fold in the hybrid plants compared with the an uncommon and potentially useful feature that did not cultivated sunflower (Table 2). V. encelioides leaves do exist in either the wild or the cultivated parent. Also, the not contain any amounts of tocopherols. size and shape of the flower head are rather specific and they look like gerbera inflorescence. The branching type was different from what is observed in wild species. 4. Discussion The appearance of non-parental phenotype is probably attributable to the non-additive interaction of some of Conventional crossing methods have sufficed to produce parental genes in the BC generation. Although data an intergeneric hybrid between cultivated sunflower elucidating events that have occurred between cultivated and V. encelioides. We analyzed morphological and H. annuus and wild Verbesina encelioides are missing biochemical data to infer the direction and extent of in the current investigation, it might be expected that backcrossing and introgression between distantly the presented findings may contribute to a molecular related species. Developing plants from intergeneric understanding of wide hybridization. cross H. annuus x V. encelioides showed many Regarding the primary cause of the HA-VERBENC phenotype features intermediate of both parents. phenotype, it could be concluded that the strongest

a) total antioxidant potential (DPPH) and ascorbate content

Parents and hybrid line DPPH, (%) ASC, (µM g DW-1)

H. annuus ♀ 89.57 ± 0.31 b 126.27 ± 1.21 b

V. encelioides ♂ 84.13 ± 2.1 a 105.83 ± 6.82 a

Hybrid line HA-Verbenc 89.91 ± 0.76 b 113.04 ± 7.40ab

LSD (P≤0.05%) 3.68 17.47

b) total phenol and flavonoid content

Phenols, Flavonoids, Vitamin E, Parents and hybrid line (mg g DW-1) (µM g DW-1) (µM g DW-1)

H. annuus ♀ 25.94 ± 0.63 b 12.93 ± 0.27 a 3.29 ± 0.88 b

V. encelioides ♂ 9.76 ± 0.36 a 17.41 ± 1.36 b 0

Hybrid line HA-Verbenc 40.70 ± 0.57d 33.62 ± 0.82d 1.87 ± 0.095 ab

LSD (P≤0.05%) 1.68 2.68 1.79

Table 2. Antioxidant content in leaves of cultivated sunflowerH. annuus, Verbesina encelioides and intergeneric line with compact plant architecture HA-VERBENC. Each value is expressed as mean ± standard deviation (n=3)

496 R.D. Vassilevska-Ivanova et al.

effects are on plant architecture and petiole length, unusual changes and compact architecture, we did branching and head flowers. Also, analysis of not observe any detrimental effect of the stature on antioxidants in the hybrid plants and both parental plant growth and development; plants are both viable species revealed a number of differences. The most and fertile, and show normal vigor. Together these striking of these differences was a significant increase characteristics make HA-VERBENC line a highly in the amount of phenols and flavonoids recovered valuable research tool with the potential to provide from the hybrid line. Thus it is entirely plausible that many insights into the phylogenic relationships in increased levels of these substances are a result of Asteraceae. Furthermore, it may have an impact on the interaction between two distantly related parental QTL cloning for agronomic traits as a suitable genetic genomes. These results suggest that analysis of population to facilitate phenotyping and map-based antioxidant levels of phenols and flavonoids may also be cloning. useful as biochemical diagnostic markers for preliminary In summary, wide hybridization of Helianthus x evaluation of hybridization events. This fact is relevant Verbesina has proven to be extremely beneficial for the to both parental species, H. annuus and V. encelioides development of new sunflower hybrids, containing genes since they contain a number of natural antioxidants such of interest from the donor species. The line could be as ascorbic acid, gluthathione, tocopherols, polyphenols introduced into sunflower breeding for seed production such as flavonoids, etc. [31,32]. management. Currently, commercial sunflowers are In consideration of the wider implications of this nearly 100% hybrids produced with the cytoplasmic work, it is clear that the intergeneric cross line has an male sterility system. We have thus illustrated the use altered intermediacy phenotype with striking changes of intergeneric hybridization to broaden the genetic in a wide range of morphological traits. Despite the variability available to modern breeding.

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