This study is an attempt to describe a conventional breeding program on Greek oregano (Origanum vulgare ssp. hirtum). First of all, a descriptor list including the most significant morphological traits was developed for the starting genetic material. The program aimed to select the elite self-plants obtained from the initial population originated from Samothraki Island, in purpose to generate the ideotype of a new cultivar, characterized by desirable agronomical traits and qualitative properties. Therefore, a pedigree intraselection method, based on widely spaced single-plant performance through honeycomb arrangement, was applied. The existing genetic/phenotypic variability of plants and families produced after selection was evaluated for plant’s growth and biomass density, type of stems and inflorescences and their components of yield potential. In addition, the effect of the mode of pollination on plants’ performance from two types of families obtained from self (SP) and open pollination (OP) was assessed. The evaluation for the components of yield potential, the composition of essential oil and carvacrol content, was detected in selected self plants, revealing genotypes with high yield potential. The target of this breeding program was the selection for high essential oil and carvacrol content and stabilization of valuable herbage components. The main selection criteria; high carvacrol and essential oil content, dry weight of leaves and flowers, ratio of leaves and flowers per stem, were subjected to statistical analysis using analytic hierarchy process, and plants were ranking according to their performance. The end-scope of this research was to recombine or even improve the starting population of local population of Greek oregano, according to mean essential oil content and carvacrol, biomass production and uniformity in desirable yield components using efficient breeding and statistical tools of selection.
Origanum vulgare L. (section Origanum) is an extremely variable and polymorphic species, due to cross pollination and plasticity (Azizi et al. 2012, 2016). O. vulgare L. subsp. hirtum (Link) Ietswaart, known as “Greek oregano”—‘rigani’-, is a typical species of Mediterranean ecosystems, growing in dry, sunny places from sea level up to 1500 m (Vokou et al. 1993). The plants are distinguished by the high density of glandular hairs and therefore the higher essential oil content, comparing to the other subspecies (Kokkini et al. 1994). Fleisher and Sneer (1982) have classified the Greek oregano chemotypes according to their essential oil composition in: carvacrol type, thymol type and both of them (carvacrol and thymol in almost equal amounts). The carvacrol chemotype determines the smell and use of the condiment oregano. Greek oregano characterized by high carvacrol content and for this reason, is considered of best quality (Fleisher and Sneer 1982). Several studies have shown a large diversity of O. vulgare chemotypes followed usually by phenotypic distinction (Vokou et al. 1993; Kokkini et al. 1994; Wogiatzi et al. 2009). In these studies a great variability in the yield of the essential oils and the pattern of their chemical constitution, and in particular of main oil constituents’ thymol and carvacrol, have been demonstrated. Oregano crops commonly use wild populations without the appropriate plant material selection (Farías et al. 2010; Torres et al. 2012; Chatzopoulou et al. 2014). For this reason a wide variety of products with different qualities of raw material and essential oils, are produced particularly in terms of carvacrol and thymol composition (Franz and Novak 2002a, b; Farías et al. 2010).
Due to the high chemical and phenotypic heterogeneity of oregano raw material, proper selection procedures are required to achieve uniformity, high biomass yield and desirable agronomical and qualitative features (Franz and Novak 1996; Chatzopoulou et al. 2014). Moreover, taking into account the market demand for homogenous raw material, selection and breeding represent a key role of the quality assurance system (Franz and Novak 1996). Especially in oregano, the extent variability of populations offers an excellent source for selection work (Bernath et al. 2006). The breeding of Medicinal and Aromatic plants (MAPs) is efficient and successful, after only few selection steps, by exploiting the high available natural variability (Pank 2007). The characterization of this high phenotypic variability observed in oregano populations, through suitable descriptors is of major importance in the field of MAPs breeding (Pank 2005). For this reason, an oregano descriptor system has been developed by European Cooperative Programme for Plant Genetic Resources (ECPGR), MAP Working Group (WG) (www.ecpgr.cgiar.org). Also, Farías et al. (2010) have reported a similar one, for characterization of oregano accessions preserved in Argentine Littoral region Germplasm bank. Moreover, Torres et al. (2012) evaluated the quantitative traits with highest discriminated value, in twelve oregano clones, and Bernath et al. (2006) demonstrated great variation concerning growth habit and production characteristics, among selected lines of O. vulgare ssp. hirtum. Franz and Novak (2002a, b) described the fundamental criteria for oregano breeding work, comprising quality characteristics: essential oil yield and high carvacrol content and certain agronomic traits; herbage yield, growth biomass, plant habit and tolerance to biotic and abiotic stress conditions.
Due to the great worldwide economic importance of oregano (Bansleben et al. 2009; Bennett 2010), and the need to improve the crop, we initiated a breeding program based on the exploitation of oregano variability in Greece. In previous works (Goliaris et al. 2002; Chatzopoulou et al. 2014), clones and native populations collected from different geographical areas of Greece, were evaluated for their genetic distances, essential oil content and the most characteristic chemical compounds (carvacrol and thymol), while the elite ones were evaluated in the same ecological conditions. Using promising genotypes as starting material and following pedigree method, we aim to develop Half Sib lines, with desirable agronomic traits and yield potential, whilst maintaining the unique high quality essential oil of the species. In PB&GRI a honeycomb breeding methodology is applied for crop improvement (Vlachostergios et al. 2011). Honeycomb methodology has been also proposed as a breeding procedure to develop cultivars that entirely meet the needs of sustainable agriculture (Fasoula and Tokatlidis 2012).
In this work, we present the results of a breeding effort on a promising Greek oregano population, and the evaluation of the favorable selected genotypes, aiming to provide the elite ones through intrapopulation on a new cultivar. Our target is furthermore, to generate the ideotype and the features of the new cultivar, characterized by desirable agronomic traits, essential oil yield and high carvacrol content using analytic hierarchy process as a statistical tool.
Materials and methods
Starting material and pre-breeding methodology
Fifty oregano plants, generated from a promising cultivated population, in terms of essential oil yield and carvacrol content (Chatzopoulou et al. 2014), initially originated from Samothraki island, were grown on rows, at 1 m distance between plants and rows, in the experimental field of PB&GRI (40°34′35″N 22°57′19″E). The species was authenticated as Origanum vulgare ssp. hirtum and a voucher specimen is deposited at the Herbarium of the Department of Medicinal and Aromatic Plants. The plants were equally and in regular basis drip irrigated and hand weeded. The soil properties at the experimental field were as follows: soil type: red loam, pH 7.71%, clay: 39.0, organic matter: 1.4%, P2O5 (ppm): 45, K2O (ppm): 520.
At the first experimental period when the plants were at the second year of growth development (Fig. 1), Mass selection was applied and thirty-four single plants expressing the following desirable agronomical traits were selected: earliness in blooming, plant growth habit, density of foliage, branching density and type of inflorescences.
A descriptor list was generated in accordance to Draft Descriptor List Origanum vulgare L., developed by the WG on MAPs of ECPGR (www.ecpgr.cgiar.org) taking into account certain morphological and agronomical characteristics, grouped into 8 classes:
Type and habitat of plant growth
Mean plant’s height (cm)
Herbage and characteristics of stem
Colour and shape of leaves
Type and characteristics of inflorescence and colour of flowers
Date of full flowering
Colour and weight of seeds
Chemical composition at full bloom
These characteristics were obtained from the most representative plants of the initial population, during full flowering in order to develop the Cultivar’s Descriptor List.
On to this experimental period, and before flowering stage, the half of inflorescences of each designated single plant, was covered with paper bags for Self Pollination, though the rest half, remained uncovered under open pollination (OP) conditions until the seeds’ maturity (Fig. 1). Seeds from self pollinated (SP) and (OP) plants were collected, and totally 11 (SP and OP) families, were generated (Fig. 1).
At the second experimental period, twenty seedlings from each of the 11 selected families *(13 genotypes) (Fig. 2) were established in a R-13 honeycomb experimental design (Fasoulas 1988; Tokatlidis 2000; Fasoula and Tokatlidis 2012) and the Pedigree intra—selection method, based on widely spaced single-plant performance, was applied (Figs. 1, 2). The observations and measurements referred to the productive characteristics: plant foliage coverage (PFC), fresh weight (FW) and dry weight (DW) per plant were estimated during the first biological cycle of oregano plants. Plant height, FW and DW per plant, were estimated during the second year of cultivation (Figs. 1, 2).
At the third experimental period, taking into consideration the main uses of oregano; spice, herbal drug and mainly the essential oil, the principal criteria for selection and breeding were defined as follows:
Essential oil content (basis on Dry Weight %),
Carvacrol content (%),
Dry weight (DW) of leaves and flowers (g)—biomass and
Ratio of leaves and flowers per stem
The final selection and ranking of self plants per family according to breeding value, was accomplished through the Analytic Hierarchy Process.
Essential oil isolation
The essential oil content was determined (a) in the initial population and (b) in individual plants, in the honeycomb trial, using a Clevenger-type apparatus according to the European Pharmacopoeia. Thirteen grams of dried plant material (leaves and flowers), were subjected to hydro distillation for 1.30 h, with a distillation rate 3–3.5 mL/min. The essential oil content was estimated on the basis of DW plant material (mL/100 g), after three replicates. The essential oils obtained were dried over anhydrous sodium sulphate and stored at 4–6 °C.
Analysis of essential oil
The essential oils were analyzed by GC-MS on a fused silica DB-5 column, using a Gas chromatograph 17A Ver. 3 interfaced with a mass spectrometer Shimadzu QP-5050A supported by the GC/MS Solution Ver. 1.21 software, using the method described previously (Sarrou et al. 2016a). The conditions of analysis were: injection temperature: 260 °C, interface heating: 300 °C, ion source heating: 200 °C, EI mode: 70 eV, scan range 41–450 amu, and scan time 0.50 s. Oven temperature program: 55°–120 °C (3 °C/min), 120–200 °C (4 °C/min), 200–220 °C (6 °C/min) and 220 °C for 5 min. Carrier gas He, 54.8 kPa, split ratio 1:30. The relative content of each compound was calculated as percent of the total chromatographic area and the results are expressed as means of three replicates.
The identification of the compounds was based on comparison of their retention indices (RI) relative to n-alkanes (C7–C22), with corresponding literature data and by matching their spectra with those of MS libraries (NIST 98, Willey) (Adams 1995).
The data (characteristics of plants development and components of yield potential) from the R13 honeycomb trial were analyzed separately for each cultivation year, within the methodological frame of Analysis of Variance (ANOVA), in order to compare 13 genotypes representing the 11 families (11 OP and 2 SP). Separate analysis per year was preferred instead of a combined analysis (repeated measurements over years), because the first cultivation year, oregano plants are developed mostly horizontally, while the second year the plants are fully developed, expressing thus their distinctive phenotype. Means’ comparisons were accomplished with the Duncan’s multiple range test. The statistical significance in all hypothesis testing procedures was predetermined at a = 0.05. All statistical analyzes were done with the statistical package SPSS v.15.0 (SPSS Inc. Chicago, Illinois, USA).
In order to rank the most effective genotypes from the honeycomb arrangement (40 plants of oregano), the analytic hierarchy process (AHP) was used, setting as principal criteria the carvacrol content (%), essential oil content (%), DW of leaves and flowers (gr), and ratio of leaves and flowers per stem. The AHP is a popular method for multiple criteria decision-making and it relies on the judgments of experts (Saaty 1987). Consequently, complex decisions could be analyzed and evaluated by means of mathematics (optimization methods) and psychology. AHP provides measures of judgment consistency, derives priorities (weights) among a number of criteria (variables) and alternatives (e.g. objects, persons, or situations), and simplifies preference ratings among decision criteria and among alternatives using a series of pair-wise comparisons. The first two steps in the AHP are crucial. The first step is to determine the criteria for rating the alternatives and the second one is to determine the relative importance of the criteria by making pair-wise comparisons (one to one) between each criterion. The second step provides the basic data (the judgment matrix D of pair-wise relative importance between criteria) used in the AHP for determining the “weights” (priority vector) of the criteria taking into account their relative importance. At the final step the alternatives are ranked according to their relative importance taking also into consideration the relative importance of the criteria used. More information about the theory, the mathematics, and the implementation of AHP can be found in Saaty (1987, 2008), and in Wikipedia (https://en.wikipedia.org/wiki/Analytic_hierarchy_process). In this study, AHP was performed in MS EXCEL enhanced with the add-in “Matrix and Linear Algebra Package for EXCEL ver. 3.2.1″.
In this study the alternatives were 40 effective plants of oregano and the criteria used were the X1 carvacrol content (%), X2 essential oil content (%), X3 DW of leaves and flowers (gr), and X4 and ratio of leaves and flowers per stem (Matrix 1).
Based on the above judgment matrix D, carvacrol content (%) has equal importance with essential oil content (%) and is 1.5 times more important than DW of leaves and flowers (gr), carvacrol content (%) is 1.5 times more important than ratio of leaves and flowers per stem, essential oil content (%) is two times more important than DW of leaves and flowers (gr), essential oil content (%) is two times more important than ratio of leaves and flowers per stem, and DW of leaves and flowers (gr), has equal importance with the ratio of leaves and flowers per stem. The elements of D below the diagonal are the reciprocals of the corresponding elements above the diagonal.
These criteria and their relative importance have been chosen because of the exploitation fields of oregano, in particular the bioactivity and pharmacological properties of oregano essential oil (Perez-Conesa et al. 2011; Jayakumar et al. 2012), which is associated to the content of carvacrol, that is affected by intrinsic and external parameters. Furthermore, to ensure the homogeneity in final product, high quality’s raw plant material is required, having high biomass production and yield of the desired constituents, suited to different market’s demands and specifications (Carlen 2016). Thus it’s becoming apparent that the standardization of the starting plant material is essential, since it is the most important factor in manufacturing herbal based products (Garg et al. 2012).
Descriptor list, growth characteristics and components of yield potential
A Descriptor list was generated in order to characterize the phenotypic traits of the starting population—Samothraki (Table 1). The promising oregano native population is characterized by erect plant growth habit, with mean plants’ height 74 cm, high branching density and stems colored dark red and green. Plants display long and dense inflorescences with white flowers and petals slightly exceed the calyx tube (Table 1).
Yield components and characteristics of oregano plants, corresponding to 13 genotypes (comprising the 11 families), were evaluated during the first and second plants biological cycles, in a honeycomb trial. Specifically, PFC, FW and DW were estimated during the first cultivation year, while plant height, FW and DW were determined, on individual plants, at full flowering, during the second cultivation year. The results showed statistically significant differences among the examined oregano families (Table 2). In general, assessment of first year’s data showed that OP families 1, 2, 5 and 11, were eminent taking into consideration the PFC, FW and DW, which ranged from 61–66% (PCF), 243–312 g (FW) and 103–120 g (DW), respectively. In the second cultivation year (Table 2) when oregano plants expressed completely their potentially productive traits, the families OP2, OP4 and OP11 exceed in plant FW and DW, while no significant differences observed in plants height. Although the main aim of breeding program is intrapopulation selection under OP conditions for the production of Half Sib families (HS), also the development of SP lines could be proved useful to determine: (a) inbreeding depression through years of selection and (b) the real potential of each population under homozygosity. Comparing the OP and SP corresponded families (SP1 and SP2), it appears that the mode of pollination significantly affected the plants’ productive characteristics, with OP plants exhibiting higher biomass production (FW and DW basis) in comparison to SP ones, in both experimentation years (Tables 2 and 3).
Based on visual-phenotype observation, during the developmental stage in the second cultivation year (Table 3), the most efficient, healthy and phenotypically superior plants (40 plants, out of totally 218 including in the honeycomb trial), according to the following criteria, were detected and further evaluated: plant growth habit, density of foliage, branching density and type of inflorescences. Self plants from OP 10 family were excluded, due to their diverging from the above criteria. Among the 10 selected OP families, plants from 2, 3, 4, 5 and 6, yielded the greatest biomass production (DW leaves and flowers) varying between 311 and 386 g per plant, while families 3 and 6 exhibited the higher number of stems per plant with the mean value 410–536, respectively (Table 3). When the effect of mode of pollination was furthermore assessed, it was observed that SP plants (Families SP1 and SP2) resulted to limited components of yield potential, while they slightly exceeded in mean length of inflorescences, compared to the corresponded OP plants (Table 3).
Essential oil and carvacrol content
The essential oil content of the 40 selected individual plants from the honeycomb arrangement, varied between 6 and 11.1% and carvacrol ranged among 52 and 87% (Fig. 3). Concerning the most significant components of yield potential, it was observed great diversity (CV 31 and 46%) for leaves and flowers (DW), ranged between 134 and 462 g. Similarly, the ratio of leaves and flowers per stems fluctuated from −1.2 to 1.
In Table 4, are presented the content and composition of the essential oil of the starting oregano population Samothraki and the 10 selected families (after 1 year of Pedigree selection through honeycomb arrangement). Twenty-one compounds were identified, accounting for the 98.62–98.87% of the total essential oil, with carvacrol, p-cymene and γ-terpinene as major ones. In addition, compounds with concentration higher than 1% such as α-thujene, β-myrcene, α-terpinene, β-caryophyllene and β-bisabolene were also identified.
As it concerns the carvacrol concentration (%) in the starting population and the selected families, although the mean value (82%) was not differentiated significantly from the starting population (81%), the selected individual plants per family indicated high carvacrol content, up to 87%, and enhanced essential oil content as well, up to 8.1% (Table 4; Fig. 3). Furthermore, selected families exhibited slightly higher hydrocarbon monoterpenes (1.55%) and limited content (2.43%) of sesquiterpenes, compared to the starting population.
Analytic hierarchy process
The AHP analysis was performed according to 4 principal selection criteria for breeding (essential oil and carvacrol content, DW of leaves and flowers and ratio of leaves and flowers per stem). The combined effect of them, classified the 40 individual plants in a decreasing order (Table 5). The superior 12 plants with the highest score weight values, ranging between 4.07 and 3.08%, belong to seven OP families (1, 2, 5, 6, 8, 10 and 11) and one SP (1) family. Consequently, the first 12 plants in the ranking list represent the highest potential for the recombination and improvement of the starting population Samothraki, exhibiting both superior qualitative; essential oil (11–8.9%) and carvacrol (86–81%) content, and agronomical traits; DW of leaves and flowers (462–169 g) and ratio of leaves and flowers per stems (1.3–0.6) (Fig. 3).
Discussion and conclusions
It has been well documented that Origanum species are characterized from high phenotypic and chemotypic diversity (Tonk et al. 2010; Kassahun et al. 2014), which is also supported from high intraspecific coefficient of variation observed in our data. Compared with other traditional food crops, breeding of MAPs is still in initial stages (Pank 2007), while the exploitation of the high available natural diversity within the species provides a significant advantage to breeders (Novak et al. 2008). This high MAPs genetic diversity, offers the opportunity to get high selection response in a relatively short time. Generally, high natural diversity within a species is one of the main reasons that conventional breeding approaches are mainly used, and they are relatively cheap methods as well (Carlen 2016). To our knowledge, limited works focusing on conventional breeding of oregano species have been reported (Langbehn et al. 2002; Bernáth et al. 2006; Simonnet et al. 2013). Plethora of the published works aimed to identify morphological and genotypic differences, through characterization the volatile metabolic profiles of different species of oregano (Andi et al. 2011; De Falco et al. 2013; Economou et al. 2014; Martínez-Natarén et al. 2014; Gonceariuc et al. 2015), while the last decade efforts have been done for the development of molecular markers for genotyping and molecular breeding (Ayanogç et al. 2006; Novak et al. 2008; Katsiotis et al. 2009; Azizi et al. 2012, 2016).
The cultivation of MAPs is strongly interconnected with sustainable agriculture and this in turn is reliant upon the availability of well-adapted cultivars that satisfy its specific requirements without compromising productivity (Fasoula and Tokatlidis 2012). Honeycomb design meet entirely the needs of sustainable agriculture, and have major principles that distinguish this method from other conventional breeding schemes and experimentation designs that includes the large interplant distance (i.e. absence of competition), which is used to minimize stress and eliminate genotype by density interaction, and the systematic entry arrangement to cope with the soil heterogeneity. Selection in the absence of competition optimizes heritability and response to selection by allowing application of high selection pressure, eliminating the confounding effects of the negative relationship between yielding and competitive ability and maximizing the phenotypic expression and differentiation (Fasoulas and Fasoula 2002). In addition, honeycomb designs ensure effective soil-heterogeneity control, to compare different entries by allocating each entry systematically and uniformly according to a hexagonal arrangement on the entire field (Fasoulas and Fasoula 1995).
Furthermore, the use of AHP statistical method provide to researchers the flexibility to regulate or select the magnitude of criteria according to the aim of experimentation, the plant species used in breeding program, and the environment of experimentation, as well.
At the beginning of a breeding program it is very important to get information about the divergence of target traits within a species and the natural populations, in order to define selection criteria and the breeding strategy. Franz and Novak (2002a, b) proposed certain selection criteria for oregano, based on the essential oil yield and depending on the use of end product; as dried herb, an amount of 1–3% of essential oil is desirable, though for essential oil production, a yield higher than 3%, is suggested. According to that, genotypes of the starting population Samothraki, evaluated in our study through the honeycomb arrangement, contain up to 8% of essential oil and high carvacrol content and could be characterized as elite ones for essential oil production. Furthermore, considering the oregano essential oil worth in the international trade, and the fact that its components represent important source of pharmacologically active compounds or agrochemicals, it becomes apparent the importance of appropriate oregano breeds, suitable for the production of high yields essential medicinal oils (Zunino and Zygadlo 2004; Torres et al. 2012). Up to date the available literature reports the existence and variability of different chemotypes of O. vulgare ssp. hirtum; carvacrol or thymol; carvacrol/thymol chemotype (Vokou et al. 1993; D’Antuono et al. 2000; Johnson et al. 2004; Economou et al. 2011; Sarrou et al. 2016b) and linalyl acetate/linalool chemotype (De Martino et al. 2009). As carvacrol is described the characteristic compound of the volatile aroma of O. vulgare ssp. hirtum, exhibiting plethora of bioactivities; antibacterial (Perez-Conesa et al. 2011), anticancer (Karkabounas et al. 2006), antioxidant (Jayakumar et al. 2012), antibiotic (Keawchaoon and Yoksan 2011), insecticide (Sedy and Koschier 2003) etc., it was one of the target traits for selection in our breeding program. Carvacrol is generally considered safe for consumption and has been approved by the Federal Drug Administration as supplementation in broiler feed and it is also included in the list of chemical flavorings of the Council of Europe, permitted in alcoholic beverages, baked goods, chewing gum, condiment relish, frozen dairy, gelatin pudding, nonalcoholic beverages, and soft candy (Botsoglou et al. 2002; De Vincenzi et al. 2004; Giannenas et al. 2005). Additionally, according to the opinion of EFSA, carvacrol is considered safe for all animal species at maximum level of 5 mg/kg complete feed when used as flavouring, and at that dose it does not pose any risk for the environment (EFSA Journal 2012).
Our investigation revealed that the intra-selection from honeycomb arrangement, lead to improved plant material, exploiting the variability of superior genotypes. Our future research, focus on the propagation of the elite individual plants, in order to investigate further the heterogeneity the upcoming years. In addition, observations and data between SP and OP plants provide the background, to evaluate the inbreeding depression through years of selection, the real potential of each population under homozygosity and seed germination difficulties under SP conditions in Oregano vulgare ssp. hirtum.
Medicinal and aromatic plants
Gas chromatography-mass spectrometry
Analytic hierarchy process
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We would like to express our gratitude and thanks towards Dr. Aggelos Markos for his invaluable help in developing the spreadsheet for running the AHP.
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Sarrou, E., Tsivelika, N., Chatzopoulou, P. et al. Conventional breeding of Greek oregano (Origanum vulgare ssp. hirtum) and development of improved cultivars for yield potential and essential oil quality. Euphytica 213, 104 (2017). https://doi.org/10.1007/s10681-017-1889-1
- Origanum vulgare
- Honeycomb design
- Morphological descriptors
- Essential oil