Turkish Journal of Agriculture - Food Science and Technology, 6(12): 1684-1689, 2018 Turkish Journal of Agriculture - Food Science and Technology Available online, ISSN: 2148-127X www.agrifoodscience.com, Turkish Science and Technology Evaluating an Interspecific Helianthus annuus × Helianthus nuttallii Line for Use in Sunflower Breeding Program Roumiana Vassilevska-Ivanova1*, Ira Stancheva2, Maria Geneva2, Zoya Tcekova1 1Department of Applied Genetics and Biotechnology, Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences Akad. Georgy Bontchev Str., Bl 21, 1113 Sofia, Bulgaria 2Department of Plant Mineral Nutrition and Water Relation, Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences Akad. Georgy Bontchev Str., Bl 21, 1113 Sofia, Bulgaria A R T I C L E I N F O A B S T R A C T Interspecific cross was made between the common sunflower inbred line HA89 and an Research Article accession of wild Helianthus nuttallii (2n=2x=34) resistant to the most harmful disease complex and tolerant to drought and high temperature. The recombinant inbred line was a Received 13 June 2017 BC1F15 progeny. The most remarkable feature observed was the vigorous plant habit that Accepted 05 June 2018 manifests itself in measurable morphological characters such as increment plant height, stem diameter and diameter of the head. The line was non-branched (monocephalic) Keywords: without anthocyanin pigmentation and possessed good agronomic characteristics. Along Antioxidants with the morphological and reproductive traits, some biochemical characteristics related Enzymes to antioxidant activity were associated to wide hybridization. The overall characteristics Gene Transfer of HA-Hnutt line make it a useful plant material for research on interspecific Pre-breeding Line hybridization in Helianthus genus. Wide Hybridization *Corresponding Author: E-mail: [email protected] DOI: https://doi.org/10.24925/turjaf.v6i12.1684-1689.1361 Introduction During the last few years, there has been an increasing In the course of our study on wide hybridization in interest in the use of wild sunflower relatives in particular sunflower, we obtained a number of recombinant inbred breeding programs which aim was providing useful lines distinguishing from both parental species. Notably, materials to support practical breeding strategies. To some of these lines could be used as a pre-breeding plant produce potential new cultivars, interspecific and material in many modern breeding programs that make intergeneric hybridizations have been performed between substantial use of wild relatives. common H. annuus and its wild relatives (Faure et al., Herewith, we report the results of interspecific 2002; Breton et al., 2012; Vassilevska-Ivanova et al., hybridization Helianthus annuus × Helianthus nuttallii, in 2013; Vassilevska- Ivanova et al., 2014; Vassilevska- particular, development of a recombinant inbred line Ivanova et al., 2015; Liu et al., 2017; Seiler et al., 2017). produced by conventional cross. The effect on antioxidant Helianthus is a diverse genus comprised of 51 species capacity as well as the content of the antioxidants such as with 14 annual and 37 perennial, all native to North ascorbate, tocopherols, phenols and flavonoids, and America (Shilling, 2006). The very narrow genetic base antioxidant enzymes was investigated. of common sunflower has been broadened by the infusion of genes from wild species, which continue to be a Materials and Methods valuable source of desirable agronomic traits. Helianthus nuttallii T. & G. (2n=2x=34) (Nuttal sunflower) was Plant Material involved in our hybridization program since it appears to The interspecific line H. annuus × H. nuttallii display an immune tolerance to Sclerotinia in USDA originated from pollination of cytoplasmic male sterile genetic stocks (Feng et al., 2007; Seiler et al., 2017), (cms) line HA89 (female), a public oilseed sunflower resistance to the most harmful disease complex (Seiler inbred maintainer line released by US Department of and Marek, 2011), and also it has shown to have drought Agriculture–Agricultural Research Service, with bulked tolerance caused by water deficit under field condition pollen from wild perennial Helianthus nuttallii (male). (Seiler et al., 2017). The population of H. nuttallii was grown from seeds Vassilevska-Ivanova et al., / Turkish Journal of Agriculture - Food Science and Technology, 6(12): 1684-1689, 2018 originally obtained from the experimental garden located measured as the increase in absorbance recorded at 470 at Bloomington (Indiana), USA. The cross was made nm. The enzyme activity was expressed as nmol H2O2 mg using the conventional hybridizing method. Each protein-1 min−1 (Plewa et al. 1991). Ascorbate peroxidase experiment was carried out on flower head that had been (APX) (EC 1.11.1.1) activity was determined measured protected from foreign pollen by bagging. First- according to the method of Nakano and Asada (1981). generation hybrid plants were verified using The concentration of oxidized ascorbate was calculated morphological and cytological methods, and F1 hybrids by the decrease in absorbance at 290 nm. Enzyme activity were back-crossed to common sunflower to obtain BC1. was quantified using the molar extinction coefficient for −1 −1 Some BC1 progeny revealed a vigorous phenotype which ascorbate (2.8 mM cm ), and was expressed as moles was considered as valuable for further breeding. These of ascorbate oxidized per milligram of protein per minute plants were selected for and fixed after the BC1 to (McKersie and Leshem 1994). Soluble protein content produce an interspecific line (namely HA-Hnutt). Seeds was determined by the method of Bradford (1976) using from advanced plant generations were produced after self- bovine serum albumin as a standard. pollination under a bag. The plants growth conditions that were employed have already been described (Vassilevska- Antioxidant Capacity Ivanova and Naidenova, 2005). The line represented here The total antioxidant capacity in fresh leaves was is F15 progeny of selfed plants; it was raised in the measured from bleaching of the purple coloured methanol Experimental Field of the Institute of Plant Physiology solution of free stable radical by the method of Tepe et al. and Genetics, Sofia, Bulgaria (located at 42º50´ N, 23º00´ (2006). Antioxidant capacity of the sample was calculated E, 595 m above the sea level) during 2011-2016. Seeds using the equation: were sown in 5 m rows spaced 0.70 cm apart with three replications. The sowings were performed in late-April. % Antioxidant activity (I) = (Ablank-Asampe/Ablank)×100 Conventional management practices were used. Where Ablank is the absorbance of the control sample Phenotypic Observations (containing all reagents except the test compound), and The morphological traits were recorded of thirty Asample is the absorbance of plant extracts. random plants for hybrid line and its parent. Methods The concentration of total phenols in the extracts was used in this investigation for hybridizing plants, fertility measured spectrofotometrically by Folin-Ciocalteu tests and morphological comparisons are the same as method and calculated as caffeic acid equivalents (Pfeffer those described in previous report (Vassilevska-Ivanova et al. 1998). The results were expressed in milligrams of and Naidenova, 2005). The following phenotype traits caffeic acid per gram of dry weight. The total flavonoids were recorded: plant height (cm), head diameter (cm), content was measured spectrofotometrically by the days to flowering (50%), days to maturity (50%), pollen method of Zhishen et al. (1999) using a standard curve staining using the acetocarmine test, thousand seeds with catechin as the standard. Total flavonoids content weight (TSW) (g), and the kernel ratio (%) using the was expressed in milligrams of catechin equivalents per equation: Kernel ratio (%) = kernel weight (g)/grain gram of dry weight. weight (g) × 100. Morphological floral characteristics included the number of ray flowers, the length of the Statistical analysis corolla of ray flowers (cm), and the width of the corolla of Data were subjected to one-way ANOVA analysis of ray flowers (cm). All floral characteristics were measured variance for comparison of means, and significant at the end of anthesis. differences were calculated according to Fisher LSD test at the 5% level using a statistical software package Antioxidant Enzyme Analyses (Statigraphics Plus, version 5.1 for Windows). Enzyme extracts were prepared by homogenizing plants tissue in a pre-chilled mortar in 20 ml chilled Results extraction buffer (pH 7.8). Extracts were then centrifuged at 12 000 g for 30 min at 5°C. Enzyme assays were Phenotype Characteristic and Agronomic Traits conducted immediately following extraction. Superoxide The morphological features of both parental genotypes dismutase (SOD) (EC 1.15.1.1) was measured by and the selected line (H. annuus × H. nuttallii) were photochemical method described by Giannopolitis and assessed (Table 1). In many respects the developing Ries (1977). Assays were carried out under illumination. hybrid line revealed intermediacy to the parental species. One unit of SOD activity was defined as the amount of At maturity, the all plants definitely distinguished in enzyme required to cause 50% inhibition of the rate of p- comparison with its parents showing vegetative vigor and nitro blue tetrazolium chloride reduction at 560 nm. increment in the growth in height and in the