Journal of Applied Botany and Food Quality 90, 106 - 114 (2017), DOI:10.5073/JABFQ.2017.090.013 1Department of Plant Anatomy and Cytology, Maria Curie-Skłodowska University, Lublin, Poland 2Department of Mathematical and Statistical Methods, Poznań University of Life Sciences, Poland 3Department of Vegetable Crops and Medicinal Plants, University of Life Sciences, Lublin, Poland 4Botanical Garden of Maria Curie-Skłodowska University, Lublin, Poland Effect of environment fluctuations on biomass and allicin level in Allium sativum (cv. Harnas, Arkus) and Allium ampeloprasum var. ampeloprasum (GHG-L) Dorota Tchórzewska1*, Jan Bocianowski2, Agnieszka Najda3, Agnieszka Dąbrowska4, Krystyna Winiarczyk1 (Received September 6, 2016) * Corresponding author Summary Climate variables such as temperature and precipitation are the major abiotic environmental factors determining the yields in crop plants. Given the observed trends in climate change, it is important to carry out analyses aimed at description and selection of plant species characterised by not only the best performance traits but also the best adaptation to climate changes. This study focused on phenological- morphological-biochemical investigations, comparing Allium sati- vum with A. ampeloprasum var. ampeloprasum GHG-L. We present analyses of economically important traits (biomass and allicin le- vels) in garlic and GHG-L grown in ecological system and effect of environment fluctuations on these traits. Comparative analysis of the biomass and allicin level in the underground part of garlic and GHG-L revealed not only substantial differences between the species and cultivars, but also great impact of the climate variables on these traits. It was found that garlic and GHG-L cultivated in adverse con- ditions, exhibited lower yielding rates, but the content of allicin was inversely proportional to the biomass. It should be emphasised that, irrespective of the climate fluctuations, GHG-L produced higher bio- mass and exhibited higher allicin level than garlic grown in the same conditions, indicating that GHG-L is well adapted to adverse climate changes. Keywords: Allium sativum; Allium ampeloprasum var. ampelo- prasum; allicin; temperature; drought; precipitation; morphological traits. Abbreviations: DAD – diode array detector; DW – dry weight; HPLC – high-performance liquid chromatography; IS – internal standard; LSDs – least significant differences; PCA – principal com- ponent analysis; s.d. – standard deviations. Introduction Changing environmental conditions are essential for the process of evolution, as they can contribute to expansion of some species and extinction of others. Seasonal fluctuations in air temperature and pre- cipitation rates are part of a natural cycle of growth and reproduction in plants. These climate variables exert an impact on the transpira- tion rate, which determines plant growth and productivity through the drought stress effect (Pelter et al., 2004). Currently, trends to- wards an increase in the mean annual temperature are observed in Europe (Christensen and Christensen, 2007) and additionally, periodic precipitation deficits, with lower water availability than plants’ requirements, are one of the most frequent environmental stresses (Bray, 1997; KalBarCzyK and KalBarCzyK, 2014). These atmospheric fluctuations can contribute to loss of crop plant yields, which is of particular importance in view of the human population growth worldwide (Bita and Gerats, 2013). Plants representing the genus Allium are widely used by humans, due to their medicinal properties determined by the presence of nume- rous sulphur compounds, especially Allium sativum (garlic) cultivars have been grown for hundreds of years at all latitudes (BloCK, 2010). One of the most important and best-known bioactive sulphur com- pounds is allicin, the content of which may account for up to 70 % of all thiosulfinates present in garlic (BloCK, 1992; lawson, 1998; ariGa and seKi, 2006). Allicin is beneficial to human health due to its antimicrobial, anticancer, antiinflammatory, antithrombotic, and antiatherosclrotic activities (BloCK, 1985, 1992; KoCh and lawson, 1996; sinGh and sinGh, 2008). The centuries-long cultivation of the species in varying environmental conditions has resulted in emer- gence of hundreds of garlic cultivars, whose traits have been con- served through vegetative propagation (simon and JendereK, 2003). Therefore, it can be stated that a majority of currently grown A. sati- vum cultivars are a result of spontaneous point mutations rather than genetic recombinations taking place via sexual reproduction (VolK et al., 2004). Interestingly, such plants differ from each other, some- times to a substantial degree, in their phenological and morphologi- cal traits (Pooler and simon, 1993; Pardo et al., 2007; VolK and stern, 2009). Clove wrapper colour, bulb size, yield, and flavour of garlic are greatly dependent on the growth environment (waterer and sChmitz, 1994; VolK and stern, 2009), which indicates high plasticity of the species in relation to the environment. Particularly noteworthy is the process of A. sativum flowering, which is extremely sensitive to changing environmental conditions. Pooler and simon (1994) showed that appropriate day length and temperature are es- sential factors initiating formation of inflorescence shoots. Similarly, other authors emphasise a significant impact of specific temperature and photoperiod regimes on the florogenesis and bulbing processes (mathew et al., 2011). Therefore, it can be concluded that garlic is a labile plant responding to varying environmental conditions espe- cially by changes in flowering processes, but the morphological cha- racteristics of vegetative organs might be affected as well. It should be noted, that the ability of species to adapt to environmental changes highly depends on genetic heterozygosity, which facilitates generative propagation. Given the advantages of sexual reproduction, which en- sures unlimited genetic fluctuations, extensive investigations are car- ried out to elucidate the causes of garlic sterility (KononKoV, 1953; noVaK, 1972; KonViCKa, 1973; etoh and simon, 2002; Kamenet- sKy et al., 2004; winiarCzyK, 2009; shemesh mayer et al., 20013, 2015). Although studies on generatively reproducing A. sativum ecotypes from Central Asia and Caucasia (etoh, 1986; etoh et al., 1988; etoh and simon, 2002) have resulted in emergence of fertile lines (inaBa et al., 1995; JendereK, 1998; KamenetsKy et al., 2003, 2005), there are no reports about a wider use of this type of plants. Furthermore, the emergence of fertile lines does not guarantee a possibility of mass production, since fertile garlic grown in a diffe- rent climate gradually loses its ability to produce seeds (etoh et al., Environment fluctuations on biomass and allicin level in garlic and great headed garlic 107 1988). Therefore, irrespective of the ongoing research on overcoming garlic sterility, it is important to search for new A. sativum cultivars, that will be best adapted to changing environmental conditions and have the best economically important traits. The climate changes observed currently in Europe reflected in a constantly increasing trend in the mean monthly temperatures and decreased precipitation rates, justify the need for systematic analy- ses, aimed at description and selection of garlic cultivars with not only the best performance traits, but also the best adaptation to cli- mate changes. So far, there have been no reports of the impact of the environment on such performance traits in garlic as yielding and the content of bioactive compounds in the underground parts of the plant. This paper focuses on the so far undescribed relationships between climatic factors, biomass growth, and allicin content in A. sativum and A. ampeloprasum var. ampeloprasum. The study was carried out on garlic cultivars commonly grown in Eastern Europe, i.e. Harnas and Arkus, and A. ampeloprasum var. ampeloprasum – Great Headed Garlic (GHG-L) described recently. GHG-L belongs to a separate clade within the genus Allium; it is phylogenetically re- lated to A. sativum, which was described by naJda et al. (2016). The analyses carried out for the first time, were performed in an ecologi- cal cultivation system in the natural environment. The investigations highlight both the climatic-phenological-morphological-biochemical relationships in garlic and GHG-L and the degree of their adaptation and productivity to changing climatic conditions. Materials and methods Plant materials and growth conditions The experimental material included garlic (A. sativum cultivars Har- nas and Arkus) obtained from Krakowska Hodowla i Nasiennictwo Ogrodnicze POLAN and A. ampeloprasum var. ampeloprasum (GHG-L – GenBank number: KT809295-KT809296) deposited in the collection of the Botanical Garden of Maria Curie-Skłodowska Uni- versity in Lublin under catalogue number 105/2013. All experiments were performed at the Botanical Garden of Maria Curie-Skłodowska University in Lublin situated in the NW part of the town, latitude 51º 16’N and longitude 22º 30’E. The terrain is highly diversified with altitudes from 217.0 m to ca. 178.0 m a. s. l. Brown soils formed on loess (33 % of fractions < 0.02 mm) with 2.72 % humus content and 7.1 alkaline pH in 1 mol KCl · dm-3 prevail in the area (Index Semi- num 2014 Hortus Botanicus Universitatis Marie Curie-Skłodowska). The experiment was established in a univariate randomised block design with 4 replications. After drying, single cloves of A. sativum (both cultivars) and GHG-L were planted in autumn 01.10.2013 and 2014. Thus, no herbicide, fungicide, or any chemical inputs were in- corporated, and manual weeding methods were used in each case before and throughout the investigated plant cultivation periods. Data on weather conditions (temperature and rainfall) prevailing during the growing seasons 2013/14/15 as well as the multiyear means (1951- 2005) are presented in Fig. 1; numerical data are presented in Tab. S1 (supplementary data). Phenology and morphology Observations of the developmental phases and morphological traits of the analysed species were carried out in the period between clove germination and anthesis of single flowers in the inflorescence. The length of the inflorescence shoot was measured from the base, and the inflorescence composition was analysed after growth and matu- ration of the analysed species (15.08.2014 and 2015). Morphological observations of the underground parts were conducted in the post- harvest period (30.07.2014 and 2015). Approximately 50 plants from each species were analysed. Macroscopic images were taken with a Nikon D300 camera equipped with an AF MICRO NIKKOR 60 mm objective. Single flowers from the inflorescences (Arkus and GHG- L) were examined under a stereoscopic microscope Olympus SZX16 equipped with a DP 72 camera. Biochemical analyses The analysis of the allicin content in the underground parts of A. sati- vum (both cultivars) and GHG-L were performed immediately after the harvest period. For evaluating the allicin content, we used the methods of lawson et al. (1991) and BaGhalian et al. (2005). This analysis was performed in the following steps: distilled water was added to 5 g of fresh cloves of A. sativum cultivar Harnas and Arkus Fig. 1: Meteorological data from 2013/2014/2015 and multiyear average (1951-2005) rainfall (mm) and temperature (oC). The meteorological station in Lub- lin Litewski Square (Index Seminum 2013, 2014, and 2015 Hortus Botanicus Universitatis Marie Curie-Skłodowska). 108 D. Tchórzewska, J. Bocianowski, A. Najda, A. Dąbrowska, K. Winiarczyk and A. ampeloprasum var. ampeloprasum (10 ml per g), mixed, kept for 10 min at room temperature, and centrifuged at 14000 rpm for 5 min. Next, 600 μl of the supernatant was added to methanol (1:1 v/v). The HPLC method with butyl parahydroxybenzoate as an internal standard (IS) was used for quantification of allicin. The 1 mg of IS corresponds to 8.65 mg of allicin. The HPLC conditions were as follows: LaChrom-Merck type equipped with a diode array detec- tor DAD (L–7450), quaternary pump (L–7100), degasser (L–7612), injection loop 20 μl, thermostat (L–7360), Rheodyne injector, steel column LiChrospher 100 RP C 18 (250 mm × 4 mm dimensions) filled with the stationary phase (dp = 5 μm). The mobile phase was methanol: water (50 % v/v) at a flow rate of 0.8 ml/min. Peaks were detected at 254 nm and recorded by a LaChrom–Merck recorder model L–7210. The content of allicin was calculated by the D–7000 HPLC System Manager program. Each sample was measured at least twice. Values were expressed as mg allicin 100g-1 DW. Statistical analysis Two-way analysis of variance (ANOVA) was performed in order to verify the zero hypotheses on a lack of differences between species, between years as well as the hypothesis on a lack of an effect of spe- cies × years interaction of the values of the observed traits, i.e. bulb size, bulb weight, clove size, clove weight, and allicin. For individual traits, mean values and standard deviations (s.d.) were calculated. Moreover, Fisher’s least significant differences (LSDs) were also estimated at the significance level α = 0.05. The results were also analysed using multivariate methods. The pos- sibility of graphic distribution of the species in the particular years of the study described by the bulb size, bulb weight, clove size, and clove weight together was obtained with the use of the principal com- ponent analysis. Principal component analysis (PCA) is a mathemati- cal procedure that uses orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components (nowo- sad et al., 2016). All the analyses were conducted using the GenStat v. 17 statistical software package. Results Climate parameters The investigations were conducted in the growing seasons of 2013/14 and 2014/15, which were characterised by substantial fluctuations in the climate variables (temperature and precipitation). The precipi- tation rates in the analysed period were considerably lower in 2015 than in 2014. The differences ranged from 80 % to 120 %, as noted in May (2014 - 175.7 mm/2015 - 101.7 mm), June (2014 - 62.7 mm/2015 - 16.3 mm), or July (2014 - 50.0 mm/2015 - 21.9 mm). The meteoro- logical data from the two analysed seasons were compared with the multiyear sum means. The observations of the weather variables in- dicate that 2014 and 2015 were warmer by 3.1 °C and 2.7 °C, respec- tively, than the multiyear temperature means. In turn, the atmosphe- ric precipitation rates in the analysed seasons were highly varied. The total precipitation sum in 2014 was by 166.5 mm higher and that in 2015 by 84 mm lower than the multiyear totals (Fig. 1). Detailed nu- merical meteorological data are presented in Tab. S1 (supplementary data). In the period of intensive biomass growth and accumulation of reserve substances in the Allium species, the average air temperature in 2014 was comparable to that in 2015, and the precipitation rate in 2014 was substantially higher than that in 2015. A comparison of the two seasons with the multiyear means revealed a growing trend in air temperature; in turn, precipitation varied widely with clear periodic droughts in 2015. Phenology analyses In this study, we analysed A. sativum cv. Harnas and Arkus as well as A. ampeloprasum var. ampeloprasum GHG-L, plants exhibiting genetic uniformity, investigated by our research team. The material used for reproduction (cloves) was considered to be of high initial quality based on participant surveys. A. sativum had been cultivated for many years in the Botanical Garden and the yields were moni- tored every year. Harnas, Arkus, and GHG-L have to be exposed to low temperatures before the growing season. Single cloves of each species were planted in autumn (2013 and 2014) and thus vernalised. In both 2014 and 2015, the first leaves appeared in spring between March 1 and 15 (1-6.03. Harnas and Arkus, 9-15.03. GHG-L). The inflorescence shoot in Harnas, Arkus, and GHG-L was formed in late May and early June in 2014 and 2015 (1.05-7.05. Harnas, 1.05-5.05. Arkus, 20.05-27.05. GHG-L). The observations of the development of the inflorescence shoot in the analysed species showed that anthesis proceeded in the period from 25.07 to 5.08 in Arkus and from 2.07 to 12.08 in GHG-L, whereas no anthesis was noted in the Harnas cul- tivar in 2014 and 2015. The development of the aboveground part of the investigated species was synchronous in 2014 and 2015 (Fig. 2). Fig. 2: Period of formation of the first leaf, inflorescence shoot, and anthe- sis in the species analysed in 2014 and 2015. A. sativum cv. Harnas (H), Arkus (A), A. ampeloprasum var. ampeloprasum GHG-L (G). Morphology analyses The comparative analysis consisted in morphological observations of the aboveground parts in terms of the foliage attitude, leaves width, initiation of the inflorescence, and formation of its components (flow- ers and bulbils). The underground part was assessed for its shape and the size of the bulb and its components – cloves and bulbils. The A. sativum cultivars, i.e. Harnas and Arkus, and GHG-L had scaly assimilating leaves, which initially did not differ in their shape and attitude. In contrast, they clearly differed in the width of the leaf blade, which was substantially broader in GHG-L from the beginning of the growth of the aboveground part. Furthermore, after emergence of a greater number of leaves, the foliage attitude in GHG-L changed from erect to semi-erect (Fig. 3a, e, i). A permanent and characteris- tic feature of genus Allium species is the amount of biomass (number of leaves) indispensable for formation of the inflorescence shoot. Prior to the emergence of the inflorescence shoot, Harnas and Arkus produced 7 leaves, while GHG-L had from 8 to 11 leaves. There were also differences in the shape of the inflorescence shoot; in A. sativum, it was initially curved, but it slightly straightened during the inflorescence maturation, reaching a length of 1000-1200 mm in Harnas and 1200-1500 mm in Arkus (Fig. 3b, f). In turn, the inflores- cence shoot in GHG-L was erect from the beginning to the end of its growth and reached a considerable length of 1500-1700 mm (Fig. 3j). The inflorescence in the Harnas and Arkus cultivars and in GHG-L was initially covered by a broad transformed bract called the spathe (Fig. 3c, g, k). After spathe dehiscence, an umbel-like inflorescence was visible; in Harnas and Arkus, it was composed of tiny single flowers and numerous bulbils (Fig. 3d, h). In contrast, GHG-L formed numerous flowers but no bulbils in the inflorescence (Fig. 3l). The perianth of the flower was green in all the analysed species. During the maturation period until the anthesis stage, it changed colour into purple in Arkus and GHG-L. In turn, there was no anthesis in Harnas Environment fluctuations on biomass and allicin level in garlic and great headed garlic 109 and the colour of the perianth remained green until the end of the vegetation period. In the analysed plants, the underground part, i.e. the bulb composed of cloves, did not differ in its shape of the base and distribution of cloves. Harnas, Arkus, and GHG-L had a flat shape of the bulb base (Fig. 4a, d, g) and radial distribution of bulb cloves (Fig. 4b, e, h). The plant differed in the bulb colour (Harnas, GHG-L – yellowish and Arkus – purple), the number of bulb cloves (Harnas 6-15, Arkus 5-8, GHG-L 3-11), as well as the presence of bulbils on the pedun- cles in GHG-L (Fig. 4g – small picture) and the absence of bulbils in the Harnas and Arkus bulbs. Tab. 1 presents a summary of the morphological traits of the analysed species. The analysis of the un- derground biomass was based on measurements of the size of the bulbs and cloves (Fig. 4a, c – bar) and the weight of the bulbs and cloves of the analysed species. The data obtained in 2014 indicate that the diameter/weight of bulbs in both Harnas (503.2 mm/36.13 g) and Arkus (437.9 mm/21.91 g) was significantly lower than that in GHG-L (821.4 mm/95.25 g). This trend was also observed in 2015 (Harnas – 436.4 mm/27.65 g; Arkus – 366.6 mm/18.82 g; GHG-L – 691.1 mm/84.65 g). Similarly, the values of the diameter/weight of cloves in the analysed Harnas and Arkus cultivars were lower than in GHG-L: Harnas – 179.8 mm/2.838 g; Arkus – 207.1 mm/3.86 g; GHG- L – 296.7 mm/18.722 g in 2014. While in 2015, Harnas – 126.0 mm/ 2.658 g; Arkus – 175.6 mm/3.817 g; GHG-L – 272.4 mm/14.064 g. The results of the analysis of variance indicated that the main ef- fects of the species and years as well as the effect of the species × years interaction were statistically significant for all observed traits (Tab. S2, supplementary data). The multidimensional analysis of the tested traits comparing the species and years in terms of bulb size, bulb weight, clove size, and clove weight simultaneously is shown in Fig. 5A, B, C, and D. Numerical data are presented in Tab. S3 (supplementary data). GHG-L produced greater biomass amounts than garlic; furthermore, the diameter/weight of bulbs and cloves was shown to be gretaer in 2014 than in 2015 in all the species. The multivariate analysis of variance (ANOVA) allowed discarding the tested hypotheses about the lack of multi-trait variability between the species, years and species × years interaction (P < 0.001). Individual traits are of different importance and have a different share in the joint multivariate variability. A study on the multivariate variabili- ty for treatments also includes identification of the most important traits in the multivariate variation of treatments. Principal compo- nents analysis PCA is a statistical tool making it possible to solve this problem. It showed that the analysed A. sativum cultivars formed separate groups in relation to GHG-L, and their variability ranges did not overlap. All traits with the exception of the clove size, which differentiated the cultivars best, were correlated with the first prin- cipal component (PC1). In turn, the clove size was correlated with the second principal component (PC2). PC1 explained as much as 97.67 % of the variability, whereas PC2 explained only 2.23 % of the total variability of the analysed morphological traits (Fig. 6). Allicin level analyses The phenological and morphological analyses were extended with biochemical assays, which revealed the allicin level in the under- ground part (cloves) of A. sativum cv. Harnas and Arkus as well as A. ampeloprasum var. ampeloprasum GHG-L grown in an ecological cultivation system. The analyses showed differences in the content of Fig. 3: Morphology of the aboveground part of a flowering plant of A. sativum cv. Harnas: a, b – inflorescence shoot, c – inflorescence partially covered by a spathe, d – inflorescence devoid of a spathe and a single flower and single topset; cv. Arkus: e, f – inflorescence shoot, g – inflorescence partially covered by a spathe, h – inflorescence devoid of a spathe and a single flower during anthesis and single topset; A. ampeloprasum var. ampeloprasum GHG-L: i, j – inflorescence shoot, k – inflorescence partially covered by a spathe, l – inflorescence devoid of a spathe and a single flower during anthesis. 110 D. Tchórzewska, J. Bocianowski, A. Najda, A. Dąbrowska, K. Winiarczyk this substance between garlic and GHG-L. Particularly noteworthy is the fact that there were differences in the allicin level between the vegetation seasons in all the analysed species. The cloves of the Harnas cultivar exhibited lower allicin content in 2014 (on average 1.262 g 100g-1 DW) than in 2015 (1.813 g 100g-1 DW). Similarly, Arkus had lower amounts of allicin in 2014 (1.081g 100g-1 DW) than in 2015 (1.335 g 100g-1 DW). The same tendency in the allicin level in cloves was noted in GHG-L (2014 – 1.343 g 100g-1 DW, 2015 – 2.097 g 100g-1 DW). The results of the analysis of variance indicated that the main effects of the species and years as well as the effect of the species × years interaction were statistically significant for the allicin level (Tab. 2 and 3). To sum up, the allicin content was lower in 2014 than in 2015 in all the analysed species. Noteworthy, GHG-L was found to be richer in allicin in comparison with the garlic culti- vars in both growing seasons. Discussion Climate variables, e.g. air temperature and atmospheric precipita- tion are major abiotic factors affecting plant growth and develop- ment. They induce ontogenetic development of plants, in particu- lar bulb plants, which can be observed upon vernalisation thereof (KamenetsKy and oKuBo, 2012). Crop plants have been observed to have relatively low adaptability to climate variables, such as periodic drought; hence, the genetic yield potential cannot be fully exploited in changing weather conditions (Bray, 1997). Therefore, the search for species able to adapt efficiently to adverse environmental condi- tions is desirable. Plants representing the genus Allium are widely distributed in cul- tivation and used as a valuable source of bioactive compounds; therefore, maintenance of the high level of yielding is extremely vi- tal. This can be achieved by production of new cultivars with better adaptation to climatic variables or by selection of existing varieties that can adapt to climate fluctuations. Since, A. sativum has lost the capability of sexual reproduction, many researchers have focused their attention on the impact of the environment on the formation of generative organs in this plant, and on the process of gametogenesis. This research is important in view of restoration of the capability of generative reproduction and, consequently, effective production of new varieties (inaBa et al., 1995; JendereK, 1998; etoh and simon, 2002; KamenetsKy et al., 2003, 2004, 2005; shemesh mayer et al., 20013, 2015). On the other hand, there is no detailed information on the relationship between the climate impact and garlic yielding and the content of bioactive substances such as allicin. These investigations focus on assessment of biomass and the level of allicin in two species, garlic and GHG, in response to adverse cli- mate changes. The A. sativum cultivars Harnas and Arkus analysed in this study are widely grown in central and eastern Europe. In turn, Fig. 4: Morphology of the underground part of A. sativum and A. ampeloprasum var. ampeloprasum. Single bulb (the site of the bulb diameter measurement - bar): a – Harnas, d – Arkus, g – GHG-L (bulbils located on a peduncle - small picture). Cross section across the bulb shows the distribution of cloves: b – Harnas, e – Arkus, h – GHG-L. Single cloves (the site of the clove diameter measurement - bar): c – Harnas, f – Arkus, i – GHG-L. Tab. 1: Morphological traits of A. sativum (cv. Harnas, Arkus) and A. am- peloprasum var. ampeloprasum (GHG-L). IS – inflorescence shoot. Morphological traits Harnas Arkus GHG-L Leaf width Narrow Narrow Broad Foliage attitude Erect Erect Semi-erect Leaves before IS (number) 7 7 8-11 Shape of IS Curved Curved Erect Length of IS (mm) 1000-1200 1200-1500 1500-1700 Flower colour Green Purple Purple Bulbils in the inflorescence Present Present Absent Bulb colour Yellowish Purple Yellowish Shape of bulb base Flat Flat Flat Distribution of bulb cloves Radial Radial Radial Number of bulb cloves 6-15 5-8 3-11 Bulb with bulbils Absent Absent Present Environment fluctuations on biomass and allicin level in garlic and great headed garlic 111 Fig. 5: Box-and-whisker diagram of the values of A bulb size (mm), B bulb weight (g), C clove size (mm), D clove weight (g); classified by the A. sativum cv. Harnas, Arkus and A. ampeloprasum var. ampeloprasum GHG-L and the years of the study. Fig. 6: Principal components analysis (PCA) – grouping coordination scheme of A. sativum and A. ampeloprasum var. ampeloprasum GHG-L cultivars in relation to morphological features. 112 D. Tchórzewska, J. Bocianowski, A. Najda, A. Dąbrowska, K. Winiarczyk the A. ampeloprasum var. ampeloprasum GHG-L is cultivated non- commercially in eastern Poland. GHG-L is phylogenetically related to A. sativum but, as we have shown recently, has higher nutritional values than garlic (naJda et al., 2016). Given the significant climatic differences in the two growing seasons, the periods of 2013/2014 and 2014/2015, the biomass and level of allicin were analysed to fo- cus more light on the relationship between climate fluctuations and biomass production among these two species. The species analysed during the two seasons were grown in an ecological cultivation sys- tem in the same area. The seasons were characterised by fluctua- tions in average temperatures and clearly different values of rainfall. Compared to the previous 54 years, higher values of the mean air temperature (by 3.1 °C) and higher mean precipitation rates (by 166.5 mm) were noted in 2014. In contrast, in 2015, the mean air temperature was by 2.7 °C higher, but particularly noteworthy, the mean precipitation rates were by 84 mm lower than the multiyear means. These data indicate a constant increase in the air temperature and occurrence of substantial periodic water shortages, which was also reported from Europe (marsz, 2005; Bita and Gerats, 2013; KalBarCzyK and KalBarCzyK, 2014). Thus, these two vegetative seasons are very suitable to compare the analyses with respect to biomass production by the analysed species. The first stage of our study was the comparative phenological analy- sis of A. sativum cv. Harnas, and Arkus and A. ampeloprasum var. ampeloprasum GHG-L. It showed that some periodic phenomena in the development of the analysed species, such as formation of the first leaf and the inflorescence shoot, took place synchronically in Arkus and Harnas. In contrast, the first leaf and the inflorescence shoot in GHG-L were formed at a later period than in garlic, and anthesis lasted longer than in Arkus. In Harnas, there was no anthe- sis stage in the inflorescence. However, no significant phenological differences were observed between the analysed cultivars in both growing seasons (2014 and 2015). Another step was the morphological analysis of the aboveground parts of the analysed plants. These observations indicate that Harnas and Arkus produce assimilating leaves with the same size, shape, and number, and inflorescence shoots with a similar shape, size, and similar constituents. In turn, GHG-L differed from garlic in its size, habit, and inflorescence composition, as it produced numerous flow- ers but no bulbils. These differences are associated with the fact that GHG-L resembles leek in its aboveground parts, with which it is phylogenetically related (as well as with garlic) (naJda et al., 2016). It can be stated that the environmental variables (temperature and rainfall) had no effect on the morphological traits of the aboveground parts of Harnas, Arkus, and GHG-L during the two analysed grow- ing seasons. It can be explained by the fact that, in the initial stages of analysed plant development, winter soil water resources were suf- ficient for effective growth of the aboveground parts and the periodic precipitation shortages did not produce a negative effect, indicating that initial water resources are important at first stages of the above- ground parts of the analysed plants. The subsequent comparative analysis of the underground parts re- vealed that such morphological traits as the shape of bulbs and dis- tribution of cloves were identical in all the analysed species, which however differed in the colour and number of cloves in the bulb. Ad- ditionally, GHG-L had characteristic bulbils arranged on peduncles, which were not found in Harnas and Arkus. Based on the analysis of the two vegetation seasons, it can be concluded that the climate fluctuations had no impact on the aforementioned features of the underground parts in Harnas, Arkus, and GHG-L, as is formed at early stage of plant formation. These observations are consistent with previous reports (VolK and stearn, 2009), whose authors conclude that such traits as the colour and shape of bulbs and arrangement of cloves in the bulb are species specific and independent of envi- ronmental parameters. It has been found that the yields determined as diameter and weight of bulbs and cloves in the analysed plants revealed differences between the cultivars Harnas, Arkus, and GHG- L. Noteworthy, the differences between Harnas and Arkus were in- considerable, whereas GHG-L was characterised by a significantly greater diameter and bulb and clove weight, compared with garlic. GHG (Great Headed Garlic, synonym “elephant garlic”) is known for the large size of its underground bulb and is cultivated at all latitudes as a garlic substitute (FiGliuolo et al., 2001; BohaneC et al., 2005; hirsCheGGer et al., 2006, 2010; lanzaVeChia, 2009; naJda et al., 2016). It should be emphasized that the economically important trait, i.e. the bulb size, ranks GHG in the first place among the plants from the genus Allium. The comparative analysis of the yielding level in the examined plants in both vegetation seasons showed noteworthy dependence. Significant differences were observed between the culti- vars. In 2015 with water shortage, all the plants produced bulbs with a smaller diameter and weight than in 2014, which implies that the unfavourable climate conditions led to a decrease in the yields of all the analysed cultivars. It should also be emphasised that, irrespective of the lower yields, GHG-L was characterised by greater biomass than that of garlic in both growing seasons and produced the highest yield. Our observations are in line with other authors, which reported that garlic yielding depends not only on the genetic traits of the spe- cies, but largely on climatic variables (waterer and sChmitz, 1994; VolK and stern, 2009; wanG et al., 2014). The yielding efficiency is especially important in crop plants, but an equally important trait in garlic is the content of bioactive com- pounds, e.g. allicin. The presented comparative analysis of this com- pound in the bulb (cloves) revealed intriguing results, which has never been reported for garlic species, ecologically cultivated in the natural environment. The level of allicin contained in the cloves differed not only between cultivars Harnas, Arkus, and GHG-L, but interesting dependence was noticed between the studied seasons, the level of allicin was higher in all the plants analysed in 2015 with seasonal droughts, although their yield was lower than that in 2014. These results are consistent with previous observations carried out in iso- Tab. 2: Mean squares from analysis of variance for allicin. Source of variation Number of degrees Mean squares of freedom Year 1 1.23343*** Species 2 1.16741*** Year × Species 2 0.05522*** Residual 24 0.00008 *** P<0.001 Tab. 3: Mean values and standard deviations (s.d.) of allicin levels (dry mass) in raw A. sativum cv. Harnas, Arkus and A. ampeloprasum var. ampeloprasum GHG-L (g 100g-1 DW). Year 2014 2015 Species Mean s.d. Mean s.d. Harnas 1.262 0.00608 1.813 0.00841 Arkus 1.081 0.00576 1.335 0.0092 Mean for cv. 1.172 1.574 GHG-L 1.685 0.00394 2.097 0.01626 Mean for all species 1.343 0.262 1.748 0.3259 LSD0.05 Year: 0.0069; Species: 0.0085; Year × Species: 0.01198 Environment fluctuations on biomass and allicin level in garlic and great headed garlic 113 lated systems, which showed a correlation between increased allicin content and decreased bulb weight, which was associated with modu- lation of the metabolism of these plants by humidity and temperature (Bloem et al., 2011). Furthermore, lee et al. (2013) have shown a correlation between the activity of γ-glutamyl transpeptidase (GGT), i.e. an enzyme involved in the metabolism of sulphur compounds and the temperature and humidity, which may be a direct factor contri- buting to the increase in the allicin content in garlic heads. Addition- ally, a close relationship between the growing conditions (fertilisa- tion) and the sulphur content in plants from the genus Allium has also been reported (wanG et al., 2014). Thus, allicin level may represent a sensitive indicator of metabolic changes upon fluctuating growth conditions. Conclusion This paper presents for the first time an analysis of economically im- portant traits in garlic and GHG-L grown in an ecological cultivation system, without human interference. The comparative analysis of the development of the aboveground and underground parts of each spe- cies included an important yield-determining element, i.e. environ- ment fluctuations during the two vegetation seasons. Analysed plants grown in adverse climatic conditions (periodic droughts) were cha- racterised by lower mass yields, but the allicin content was inversely proportional to the increase in biomass. In the adverse soil moisture conditions in 2015, garlic cv. Harnas exhibited a substantial increase in the level of allicin contained in the underground bulbs. It should be emphasised that, irrespective of the climate fluctuations, GHG-L had a significantly higher biomass and a higher level of allicin than garlic grown under the same conditions. The correlations between the bulb size and the allicin content in Harnas, Arkus, and GHG-L in both growing seasons are presented in Fig. 7. As already reported in earlier studies and according to the data presented in this paper, it can be concluded that A. ampeloprasum var. ampeloprasum – GHG-L represents much better characteristics from the agricultural and biochemical/nutritional point of view. Thus, greater populari- sation of GHG and introduction thereof to large-scale cultivation should be considered as an alternative to traditional garlic, especially in regions with significant climate fluctuations. 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Address of the corresponding author: Dorota Tchórzewska, Department of Plant Anatomy and Cytology, Maria Curie-Skłodowska University, Akademicka 19 Street, 20-033 Lublin, Po- land. E-mail: dorota.tchorzewska@poczta.umcs.lublin.pl © The Author(s) 2017. This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative- commons.org/licenses/by-sa/4.0/). Supplementary material I Tab. S1: Meteorological data from 2013/2014/2015 and multiyear average (1951-2005) rainfall (mm) and temperature (oC). The meteorological station in Lublin Litewski Square (Index Seminum 2013, 2014, and 2015 Hortus Botanicus Universitatis Marie Curie-Skłodowska) 3     Meteorological data from 2013/2014/2015 and multiyear average (1951-2005) rainfall (mm) 74   and temperature (oC). The meteorological station in Lublin Litewski Square (Index Seminum 75   2013, 2014, and 2015 Hortus Botanicus Universitatis Marie Curie-Sk�odowska) 76   77   Month Decade Rainfall (mm) Temperature (oC) 2013/14 2014/15 Long-term 2013/14 2014/15 Long-term 1 0 0 19.4 7.5 11.1 9.8 2 4.4 1 14.6 9.3 13.6 8.2 X 3 1 24.6 12.3 13.3 4.2 6 1 56.6 2.6 13.7 8.9 9.9 4.7 2 5.4 13.7 12 4.1 4.8 2.2 XI 3 18 6.4 11.4 3.2 -1.6 0.6 1 13 3.9 9.9 0 -2.6 -0.6 2 3.2 19.9 9.9 0.7 4.2 -1.5 XII 3 0 32.7 13.9 3.8 -1.2 -2 1 18.5 24.6 7.3 4 -1.3 -3.5 2 45.2 4.9 6 -0.4 3.2 -3.7 I 3 7.1 17.3 8.3 -1.4 1 -3.7 1 4.4 2.9 7.7 -1.5 -1.4 -3.3 2 7.7 0 9.1 3 0.1 -2.8 II 3 0.2 4.5 8.1 2.7 4.5 -2.1 1 3.8 9.9 8.4 3.3 3.4 -0.7 2 19.7 15.7 7.4 6.2 3.8 0.4 III 3 24.9 30.2 10.1 8.8 6.5 3 1 3.2 12.7 14.2 7.4 4.4 5.9 2 15.3 2.8 12.3 7.9 8.9 6.9 IV 3 26.2 25.3 14.1 14.1 12.5 9.6 1 32.7 26.8 16.6 10.6 13.1 11.6 2 88.4 8.9 18.3 12.9 13.4 13.6 V 3 72.5 76.2 23.5 17.4 13.3 13.7 1 11.2 11.1 20.8 17.4 19.1 16 2 3 0.1 21.2 16 17.9 16.3 VI 3 63.9 0 23.8 15.4 17.1 17.1 1 14.2 0.4 23.5 19.4 21.4 17.4 2 38.4 23.5 25.7 19.8 19.8 18.2 VII 3 30.6 19.7 29 21.8 20.7 18  78   Table S2 79   II Supplementary material 4     Mean squares from analysis of variance for the observed traits of the studied species 80   Trait Source of variation Species Year Species × Year Residual d.f. 2 1 2 335 Bulb size m.s. 7456640*** 1342025*** 68301*** 6413 d.f. 2 1 2 274 Bulb weight m.s. 297118.6*** 8660.8*** 974.5* 407.9 d.f. 2 1 2 592 Clove size m.s. 956818*** 258135*** 13966** 2308 d.f. 2 1 2 692 Clove weight m.s. 11790.41*** 288.64*** 363.45*** 22.32 * P<0.05; ** P<0.01; *** P<0.001 81   82   Table S3 83   Mean values and standard deviations (s.d.) for the observed traits of A. sativum cv. Harnas, 84   Arkus, and A. ampeloprasum var. ampeloprasum GHG-L 85   86       Bulb size Bulb weight Year 2014 2015 2014 2015 Species Mean s.d. Mean s.d. Mean s.d. Mean s.d. Harnas 503.2 73.89 436.4 51.73 36.13 13.52 27.65 8.17 Arkus 437.9 72.2 366.6 58.06 21.91 11.11 18.82 7.52 GHG-L 821.4 147.83 691.1 162.59 95.25 34.57 84.65 35.48 LSD0.05 S: 14.58; Y: 11.52, S×Y: 21.00 S: 3.07; Y: 2.425, S×Y: 4.423   Clove size Clove weight Year 2014 2015 2014 2015 Species Mean s.d. Mean s.d. Mean s.d. Mean s.d. Harnas 179.8 62.81 126.0 36.37 2.838 1.479 2.658 1.351 Arkus 207.1 39.41 175.6 25.38 3.86 1.964 3.817 1.843 GHG-L 296.7 65.77 272.4 52.61 18.722 11.673 14.064 8.873 LSD0.05 S: 8.73; Y: 6.9, S×Y: 12.58 S: 8.73; Y: 6.9, S×Y: 12.58 87    88   89   Tab. S2: Mean squares from analysis of variance for the observed traits of the studied species Tab. S3: Mean values and standard deviations (s.d.) for the observed traits of A. sativum cv. Harnas, Arkus, and A. ampeloprasum var. ampeloprasum GHG-L