Genetic improvement of Citrus fruits: New somatic hybrids from Citrus sinensis (L.) Osb. and Citrus limon (L.) Burm. F.

Food Research International 48 (2012) 284–290

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Genetic improvement of Citrus fruits: New somatic hybrids from Citrus sinensis (L.) Osb. and Citrus limon (L.) Burm. F. Loredana Abbate a, Nicasio Tusa a, Sergio Fatta Del Bosco a, Tonia Strano b, Agatino Renda b, Giuseppe Ruberto b,⁎ a b

Istituto del CNR di Genetica Vegetale, Corso Calatafimi, 414 90100 Palermo, Italy Istituto del CNR di Chimica Biomolecolare, Via Paolo Gaifami, 18 95216 Catania, Italy

a r t i c l e

i n f o

Article history: Received 16 February 2012 Accepted 27 April 2012 Keywords: Citrus sinensis Citrus limon Somatic hybrid Cybrid Peel essential oil

a b s t r a c t Three new somatic hybrids, namely an allotetraploid hybrid and two cybrids (2n and 4n), have been obtained by protoplast fusion of ‘Valencia’ sweet orange (Citrus sinensis L. Osbeck)+‘Femminello’ lemon (C. limon L. Burm.). The chemical composition of the essential oils of the hybrids and their parents has been studied by gas chromatography (GC) combined with a flame ionisation detector (FID) and a mass spectrometry (MS). In all, 87 components were fully characterised and grouped in four classes (monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpenes, and others) for an easier comparison of all oils. A statistical treatment by linear discriminant analysis of the compositional data from GC analyses was also carried out. The allotetraploid hybrid and both cybrids show an intermediate essential oil profile with respect to those of both parents. The contribution of ‘Femminello’ lemon parent is in all cases predominant in the production of the volatile profiles of the new hybrids; however, different behaviour in the peel essential accumulation between the allotetrapolid hybrid and the two cybrids is observed. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Citrus is the most important fruit tree crop in the world, with an annual production of approximately 123 million tonnes in 2010. The Citrus genus includes several important species with the most relevant, on a worldwide basis, being orange: 56% of total; tangerines, mandarins and clementines: 17%; lemons and limes: 11%; and grapefruit (with pomelos): 6% (FAO, 2012). Although citrus fruits are principally consumed as fresh fruit or processed juice, citrus have a great economic value being their essential oils exploited in a variety of industrial fields including beverages and food sector, as well as in the production of detergents, soaps, perfumes and cosmetics. Furthermore, numerous biological activities have been ascribed to these essential oils (Adorjan & Buchbauer, 2010; Tranchida, Bonaccorsi, Dugo, Mondello, & Dugo, 2012). Citrus species are currently submitted to intensive breeding programmes in order to produce new valuable hybrids with improved fruit quality and resistance to biotic and abiotic stresses. One approach to the genetic improvement of citrus has been through the generation of somatic hybrids, plant expressing new traits and improved characters as the result of an additive protoplast–cell fusion process, called somatic hybridization (Grosser, Ollitrault, & Olivares-Fuster, 2000). Somatic cell

⁎ Corresponding author. Tel.: + 39 0957338347; fax: + 39 0957338310. E-mail address: [email protected] (G. Ruberto). 0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2012.04.007

hybridization through protoplast fusion is a proven and useful tool in the improvement of Citrus species and varieties. This technique allows to circumvent the reproductive barriers in citrus through the transfer of entire or partial genomes between related and unrelated species (fusion parents) and lead up to a generation of new population of plants (somatic hybrid) showing novel arrangement of nuclear, chloroplast and/or mitochondrial genomes (Grosser & Gmitter, 1990; Tusa, Grosser, & Gmitter, 1990). In the last two decades a large number of intergeneric and interspecific somatic hybrids of Citrus have been created; their use in scion and rootstock improvement is under investigation in several field trials (Grosser & Gmitter, 2008, 2011; Grosser et al., 2000; Guo & Deng, 2001). In several somatic hybridization experiments, unexpected diploid plants that are phenotypically similar to one fusion parent were recovered. Molecular characterization confirmed their origin as ‘cybrid’, fusion-product containing the nuclear genome of one parent and recombinant mitochondrial genome (Guo, Cai, & Grosser, 2004). The mitochondrial DNA plays a key role in synthesis of biomolecules (carbohydrates, lipids, amino acids, vitamins and phytormones) and the generation of adenosine triphosphate (ATP). The production of cybrids is, therefore, of great interest in Citrus genetic and breeding in order to understand the role of cytoplasm during growth and the importance of cytoplasmic inheritance in Citrus improvement (Bassene et al., 2008; Sheahan, Rose, & McCurdy, 2007). One of the major goals of somatic hybridization in varietal improvement is obtaining plants bearing fruits of original and good sensory characteristics. The aromatic quality of Citrus somatic hybrids has been studied

L. Abbate et al. / Food Research International 48 (2012) 284–290

in the last 10–12 years. A study on leaf volatile compounds of seven citrus somatic allotetraploid hybrids suggested that complex forms of dominance originating from the genome of one of the two parents determine, to some extent, the biosynthesis pathways of some of the volatile compounds (Gancel et al., 2003); another study, conducted on three diploid Citrus cybrids, suggested that the main information for volatile compounds biosynthesis is contained in the nucleus but nucleo-cytoplasmic interactions strongly occurred (Fanciullino et al., 2005). It is of interest to determine if, irrespective of chromosomal status and extent of genomic asymmetry, the relationship between the somatic hybrids, the cybrids and their parents could be monitored by comparison of their secondary metabolites. As a final remark, it is worthwhile to establish if somatic hybridization or cybridization in citrus could be a useful strategy in order to enhance oil quality. Indeed, the somatic hybrid ‘V + F’ and the two Femminello cybrids (Fig. 1) here examined, have been obtained in a effort to combine the excellent fruit quality and characteristics of ‘Femminello’ lemon with the cold hardiness and tolerance to Phoma tracheiphila infections of ‘Valencia’ sweet orange. The presence of appropriate aromatic features in the resulting genotypes must be a fundamental requisite for their future use. We report the results of the analysis of the essentials oil from mature fruits of two lemon cybrids (one diploid and the other tetraploid), and one allotetraploid (4n) somatic hybrid of sweet orange + lemon. All plants were obtained during somatic hybridization experiments between protoplast of the ‘Valencia’ variety of the sweet orange (Citrus sinensis L. Osbeck), and protoplast of the ‘Femminello’ variety of the lemon (Citrus limon L. Burm. F.). The essential oil profiles of all hybrids have been compared with those of their parents.

285

large somatic embryos developing from recovered hybrid calli were grown according to the literature; cotyledonary embryos were therefore induced to develop shoots and roots into an appropriate medium. The regenerated putative cybrid plants were selected on the basis of their morphological similarities to the leaf donor parent and characterised by isozyme and restriction fragment length polymorphism (RFLP) analysis (Grosser, Gmitter, Tusa, Recupero, & Cucinotta, 1996). 2.2. Plant material Fruits of 2n cybrid, 4n cybrids and Valencia + Femminello somatic hybrid were collected at the end of February 2006 and in early March 2007. Fruits of ‘Femminello’ lemon and ‘Valencia’ orange were collected at their ripe stage, which was monitored throughout the measurement of fruit size, total soluble solids (TSS) and titratable acidity evaluation, in February and May 2007, respectively. All plants were cultivated in the experimental field of the IGV-CNR, Lascari, Palermo. 2.3. Isolation of essential oils Fresh rind tissue (flavedo, 100 g) of each sample was subjected to hydrodistillation until there was no significant increase in the volume of oil collected (3 h). The oils, whose average yield is the following: ‘Femminello’ lemon 0.9% v/w, ‘Valencia’ orange 1.2%, 2n Cybrid 1.1%, 4n Cybrid 1.0%, Valencia + Femminello hybrid 0.8%, were dried over anhydrous sodium sulphate and stored under N2 in a sealed vial at −20 °C until required, normally from few days to a week. 2.4. Gas chromatography (GC) of essential oils

2. Materials and methods 2.1. Hybridization procedures Protoplast cultivation and plant regeneration via somatic hybridization in Citrus are already standardised (Grosser & Gmitter, 1990). Plants were obtained by somatic fusion of protoplasts of ‘Femminello’ lemon isolated from leaves of young nucellar seedlings and protoplasts of ‘Valencia’ sweet orange isolated from suspension culture initiated from nucellus-derived embryogenic callus. Protoplasts of ‘Femminello’ and ‘Valencia’ were purified, mixed and fused using polyethylene glycol (PEG) method. Fusion cultures, embryogenic colonies, embryoids and

Essential oils were analysed in the fast mode on a Shimadzu gas chromatograph, model 17-A equipped with a flame ionisation detector (FID), operating software Class VP Chromatography Date System version 4.3 (Shimadzu Co., Kyoto, Japan). Analytical conditions: SPB5 capillary column (15 m × 0.10 mm × 0.10 μm) with helium as carrier gas; injection in split mode (1:200); injected volume of 1 μL (25 μL of oil in 400 μL of CH2Cl2); and injector and detector temperatures of 250 and 280 °C, respectively; linear velocity in column at 51 cm/s. The oven temperature was held at 60 °C for 1 min, then programmed from 60 to 280 °C at 10 °C/min. Percentages of compounds were determined from their peak areas in the GC-FID profiles. A second GC-FID analysis was carried out on a Hewlett–Packard (now Agilent Co., California) gas-chromatograph mod. 5890 with the following analytical conditions: Innowax capillary column (30 m×0.25 mm×0.25 μm) with helium as carrier gas; injection in split mode (1:50); injected volume of 1 μL (25 μL of oil in 400 μL of CH2Cl2); injector and detector temperatures of 250 and 280 °C, respectively; linear velocity in column at 25 cm/s. The oven temperature was held at 50 °C for 3 min, then programmed from 50 to 100 °C at 3 °C/min, and from 100 to 250 °C at 5 °C/min. 2.5. Gas-chromatography–mass spectrometry (GC–MS) of essential oils

Fig. 1. Cybrids (2n and 4n), and V + F hybrid from ‘Femminello’ lemon and ‘Valencia’ orange.

GC–MS was carried out in the fast mode on a Shimadzu GC–MS mod. GCMS-QP5050A, operating software GCMS solution version 1.02 (Shimadzu). Ionisation voltage in electronic impact mode was 70 eV, with electron multiplier at 1000 V, transfer line temperature at 280 °C, injection in split mode (1:96), and constant linear velocity in column at 50 cm/s. Analytical conditions with the apolar capillary column were the same as GC. A second gas chromatography–mass spectrometry (GC–MS) was carried out on a Hewlett–Packard (now Agilent Co., California) gas-chromatograph mod. 5890 connected to a Hewlett–Packard mass spectrometer model 5971A, with ionisation voltage in electronic impact mode at 70 eV, electron multiplier at 1700 V, and ion source temperature at 180 °C; mass spectra data were acquired in the scan mode in m/z range 40–400. Analytical conditions with the polar capillary column were the same as GC.

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2.6. Identification of essential oil components The identity of components was based on their retention indexes relative to C9–C22 n-alkanes (Alltech Italy) on the SPB-5 and Innovax columns, computer matching of spectral MS data those from NIST MS 107 and NIST 21 libraries (NIST, 1998), the comparison of the fragmentation patterns with those reported in literature (Adams, 2001) and, whenever possible, co-injections with authentic standards, which were purchased from Aldrich Chemical Co., Extrasynthese, France, and Fluka Chemie AG, Switzerland. 2.7. Statistical analysis SPSS software, version 14.1, was used to carry out statistical analysis of the data. ANOVA and HSD Tukey's test were applied to the data to determine significant differences between the analysed components; the model was statistically significant with a value of p ≤ 0.01. Multivariate analyses using stepwise discriminant analysis were carried out to estimate the contribution of parents to the hybrid essential oil composition. 3. Results and discussion Table 1 lists the composition of the essential oils of ‘Femminello’ lemon, ‘Valencia’ orange and their hybrids coming from the somatic fusion. In total, 87 components were fully identified covering more than 98% of the total composition. For an easier comparison of the oils the components were grouped into four classes: monoterpene hydrocarbons with 14 components; oxygenated monoterpenes, the most numerous class with 33 compounds; sesquiterpenes and others, with 23 and 17 compounds, respectively. Monoterpenes, both hydrocarbons and oxygenated, were the most highly represented classes: the former with a range of 76–97% and the latter with a range of 2–20%. The sesquiterpene and other classes were in all cases the least represented. The traditional way of isolating Citrus essential oils is cold pressing of Citrus peels, however, distillation and hydrodistillation are also used as alternative methods (Ferhat, Meklati, & Chemat, 2007). Both methodologies present some disadvantages: in the pressing procedure the continuous emulsion with water produces the loss of some oxygenated components (i.e. citral and alcohols), furthermore some undesirable transformations, such as hydrolysis and oxidations, may occur. On the other side, the high temperature of distillation procedure can introduce some chemical modification and the partial loss of the most volatile components. We have chosen to use hydrodistillation in order to uniform the extraction procedure, to obtain comparable yields from all samples, and to avoid the presence of non‐volatile components, which depend on the species range between 1 and 15% of the total oil. The ‘Femminello’ essential oil is characterised by a relatively low amount of limonene and a high content of β-pinene and γ-terpinene, if compared with other Citrus essential oils (Fig. 2). Another peculiar aspect of lemon oil is the high content of two couples of oxygenated monoterpenes, namely neral and geranial (known as citral) and the corresponding alcohols nerol and geraniol, being the first couple present, normally, at higher concentration (Fig. 3). It should be noted that it is the presence of these oxygenated components that makes this oil particularly appreciated, determining, also, its commercial value (Tranchida et al., 2012). The ‘Valencia’ oil is, instead characterised by ca. 92% of limonene, as usually observed in an orange essential oil. The content of oxygenated monoterpenes is very low (ca. 3%), linalool being the main component (Tranchida et al., 2012). Concerning the essential oil of the allotetraploid somatic hybrid (‘V + F’) a more marked similarity with ‘Valencia’ parent is observed even though analysing the compositional detail an intermediate behaviour, between both parents, is observed. In fact, concerning the main components, namely limonene, γ-terpinene and β-pinene, belonging to the monoterpene hydrocarbons class, their amount in

the new hybrid can be considered intermediate between those of ‘Femminello’ and ‘Valencia’ parents (Fig. 2). The content of oxygenated monoterpenes is, instead lower (below 2%) than those of both parents (Fig. 3), the total being distributed on twenty components. A different picture emerges from the analysis of the essential oil data of the two cybrids. The salient feature is the dominant role of the ‘Femminello’ lemon in the oil production of both cybrids, as well as the apparent marginality of the ‘Valencia’ orange. This dominance is, however, more marked in the 2n rather than in 4n cybrid. Considering the main classes of components and their most important constituents, both hybrids present a slightly higher limonene content than lemon, whereas the other two main compounds, namely βpinene and γ-terpinene, are more markedly present in both hybrids (Fig. 2). Concerning the oxygenated monoterpenes, the 2n cybrid contains a double amount of these components with respect to 4n cybrid, ca. 10 vs. ca. 5%, this large difference being essentially due to the different contribution of the two couples of components (neral/ geranial — nerol/geraniol) previously cited for the lemon parent (Fig. 3). These four components, in fact, amount to ca. 7% in 2n cybrid, against the ca. 3% in the 4n. Finally, the dominance of ‘Femminello’ is also confirmed by the profile of the minor components, most of them below 1% (Table 1). Statistically significant differences were found between the average content of all classes in the five samples by ANOVA and Tukey's multiple range test (Table 1). However, in order to obtain best differentiation of all fruits involved in this study, namely parents, cybrids and hybrid, all components of each essential oil were investigated by means of multivariate analysis, applying the linear discriminant analysis (LDA), which has been successfully applied elsewhere in the differentiation of citrus juices as well as peel and leaf citrus oils (Moshonas & Shaw, 1997; Rapisarda, Pannuzzo, Romano, & Russo, 2003; Ruberto, Biondi, Rapisarda, Renda, & Starrantino, 1997a; Sentandreu, Izquierdo, & Sendra, 2007; Shaw, Goodner, Moshonas, & Hearn, 2001). The graphic representation of the variables (all essential oil components) in the two functions, 1 and 2, is shown in Fig. 4. In particular, the eigenvalue associated to the first function contributed 79.3% of the variance, and the eigenvalue associated to the second function contributed 15.5% of the variance, therefore, the combination of the first and the second function gives almost 95% of the total variance of the system. Table 2 reports a list of the components with the highest statistical weight and therefore the importance of each variable (essential oil component) for the two functions. The graphic representation in the two functions shows the expected large differentiation between ‘Valencia’ orange and ‘Femminello’ lemon. The somatic hybrid ‘V + F’ is placed between parents according to the two functions, being closer to the ‘Femminello’ parent, as observed in a previous study dealing with the polyphenol profiles (Tusa, Abbate, Renda, & Ruberto, 2007). Both 2n and 4n cybrids, appear very similar, placing on an almost superimposable position with respect to the ‘Femminello’ lemon parent regarding the most important function 1, clearly confirming the strong contribution of this parent in the elaboration of the peel essential oil of both cybrids. According to the second function of the discriminant analysis, the two parents show a higher similarity and the cybrids are placed in an intermediate position of parents, whereas the allotetraploid somatic hybrid ‘V + F’, is placed fairly distant from both parents. Unfortunately, the biomolecular studies on the secondary metabolic profile, in general, and on volatile components, in particular, of Citrus somatic hybrids are rather scarce (Alonzo, Fatta Del Bosco, Palazzolo, Saiano, & Tusa, 2000; Fanciullino et al., 2005; Fatta del Bosco, Palazzolo, Scarano, Germanà, & Tusa, 1998; Gancel, Ollé, Ollitrault, Luro, & Brillouet, 2002; Gancel et al., 2005a, 2005b), therefore it is difficult to find common aspects in the accumulation mechanism of these components at the moment. In fact, previous studies on the volatile compounds from leaves and peels of interspecific somatic hybrids

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Table 1 Chemical composition of ‘femminello’ lemon, ‘valencia’ orange, V + F hybrid, 2n and 4n cybrids, essential oilsa. Peak #

RIb

RIc

Compound

‘Femminello’ lemon

‘Valencia’ orange

V + F hybrid % (± SD)

2n cybrid

4n cybrid

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74

801 802 857 932 939 955 978 982 986 993 1004 1007 1014 1021 1040 1033 1043 1065 1071 1072 1068 1090 1100 1104 1107 1119 1121 1124 1140 1142 1151 1158 1166 1166 1170 1176 1183 1184 1197 1200 1211 1212 1223 1227 1230 1236 1246 1249 1261 1265 1277 1273 1291 1292 1302 1307 1306 1351 1354 1365 1383 1394 1406 1408 1413 1424 1433 1439 1458 1458 1457 1462 1484 1492

802 1016 1060 998 999 993 1047 1115 1099 1343 1166 1292 1191 1178 1204 1241 1257 1250 1441 1464 1453 1283 1560 1396 1414 1432 1683 1640 1631 1355 1507 1479 1812 1779 1698 1667 1681 1599 1697 1553 1499 1567 1688 1623 1760 1790 1719 1674 1832 1795 1723 1750 1971 2154 2191 1604 1755 1768 1737 1768 1743 1599 1695 2051 1578 1588 1626 1719 1663 1659 1670 1658 2220 1698

Octaned Hexanald trans-2-Hexenal α-Thujened α-Pinened Camphened Sabinened β-Pinened 6-Methyl-5-hepten-2-one Myrcened Octanald α-Phellandrene p-Mentha-1(7),8-diene α-Terpinened Limonened cis-β-Ocimene trans-β-Ocimene γ-Terpinened trans-Linalool oxide (furanoid) trans-Sabinene hydrated Octanold α-Terpinolened Linaloold Nonanald cis-Thujone trans-Thujone Dehydro-sabina ketone cis-p-Menth-2-en-1-ol cis-p-Mentha-2,8-dien-1-ol trans-Limonene oxide Camphord Citronellald β-Pinene oxide cis-Chrysanthenol Borneold n-Nonanold trans-p-Mentha-1(7),8-dien-2-ol Terpinen-4-old α-Terpineold Estragoled Decanald Octanol acetated trans-Carveol cis-p-Mentha-1(7),8-dien-2-ol Citronellol Nerold Carvone Nerald Geraniold trans-2-Decenald Geraniald Perillaldehyded Limonen-10-ol Thymold Carvacrold Undecanald Undec-10-en-1-al α-Terpinyl acetate Citronellyl acetate Neryl acetate Geranyl acetate β-Elemene Dodecanald N-Methyl anthranilate cis-α‐Bergamotene β-Caryophyllened β-Copaened trans-α-Bergamotene cis-β-Farnesene α-Humulened trans-β-Farnesene β-Santalene Germacrene D Valencened

t 0.06 (0.002)

t 0.02 (0.002) 0.02 (0.003)

0.02 (0.004)

0.03 (0.004)

0.01 (0.011)

0.20 0.98 0.02 1.56 3.15

0.33 (0.043) 1.51 (0.172) 0.06 (0.010) 1.24 (0.356) 10.44 (2.197) 0.02 (0.008) 1.33 (0.041) 0.19 (0.022) t

0.32 1.50 0.06 0.89 9.22

0.25 (0.016) 71.21 (1.547)

0.27 (0.005) 0.22 (0.011) 0.07 (0.010)

0.27 (0.024) 64.24 (3.706) 0.02 (0.015) 0.14 (0.015) 8.64 (0.697) 0.02 (0.002) t 0.04 (0.004) 0.44 (0.029) 0.42 (0.042) 0.20 (0.019)

0.02 (0.001) 0.02 (0.001) 0.02 (0.001)

0.03 (0.004)

0.03 (0.003)

0.04 0.10 t 0.04 0.02 0.05

0.03 (0.014) 0.07 (0.030) 0.02 (0.002)

(0.005) (0.007) (0.005)

0.50 0.87 0.03 0.03

(0.052) (0.109) (0.004) (0.006)

0.22 1.01 0.05 0.71 7.29 0.05 1.12 0.20 t

(0.001) (0.009) (0.001) (0.007) (0.051) (0.001) (0.010) (0.004)

0.28 (0.004) 59.75 (0.369) t 0.07 (0.001) 5.65 (0.029) t 0.02 0.38 1.14 0.31 0.08 0.02

(0.000) (0.004) (0.010) (0.009) (0.002) (0.001)

0.38 (0.004) 0.35 (0.025) 0.06 (0.006) 1.51 (0.017) 1.42 (0.070) t 0.07 (0.003) 0.02 (0.003) 91.51 (0.388) t 0.07 (0.007) 0.10 (0.012) t 0.26 0.03 1.78 0.07

(0.030) (0.0029) (0.187) (0.004)

0.02 (0.002) 0.05 (0.007) 0.14 0.35 t 0.07 0.05

(0.001) (0.004)

0.10 (0.006) t

(0.002) (0.011) (0.001) (0.054) (0.086)

1.64 (0.005) 0.07 (0.004) 0.02 (0.002) 0.18 (0.004) 84.56 (0.224) 0.07 (0.006) 4.65 (0.061)

0.01 (0.000) 0.11 (0.012)

(0.001) (0.001) 0.02 (0.001)

0.82 1.41 0.30 0.06 0.01 0.02 0.03

(0.010) (0.018) (0.010) (0.001) (0.000) (0.000) (0.001)

2.39 (0.071) t 4.64 (0.062) 1.60 (0.022) 6.44 (0092)

0.11 (0.017) 0.24 (0.015) 0.43 (0.028)

0.10 0.05 0.22 0.05 0.01 0.24 0.05 0.15

(0.005) (0.021) (0.009) (0.002) (0.001) (0.013) (0.004) (0.006)

0.03 (0.004)

t

t (0.000) (0.006) (0.004) (0.007)

0.02 (0.000) 0.11 (0.005) 0.13 (0.002)

0.07 (0.001)

0.17 0.20 0.01 0.09 0.04

(0.006) (0.004) (0.000) (0.006) (0.003)

0.14 (0.009) 0.01 (0.001) 0.14 (0.011)

0.02 (0.001) 0.04 (0.003)

0.02 (0.001) 0.01 (0.000)

0.21 (0.005) 0.03 (0.001) t t

(0.021) (0.004) (0.006) (0.002)

0.08 (0.012)

0.09 (0.003) 0.14 (0.006)

0.02 0.55 0.27 0.04

0.32 0.09 0.04 0.04

0.02 0.07 0.22 0.10 0.01 0.02

(0.001) (0.002) (0.014) (0.007) (0.001) (0.002)

0.04 (0.001) 0.14 (0.006) 0.03 (0.002) 0.03 (0.003)

t t 0.06 (0.006)

0.01 (0.000) 0.07 (0.007)

(0.022) (0.008)

(0.051) (0.213) (0.007) (0.136) (1.980)

1.44 (0.017) 0.05 (0.054) 0.03 (0.014)

0.16 (0.010) 8.49 (0.736)

0.02 0.44 0.25 0.10 0.01

T 0.03 0.03 0.35 0.43 0.10 0.03

(0.009) (0.018) (0.081) (0.033) (0.007)

(0.006) (0.003) (0.066) (0.117) (0.004) (0.013)

0.01 (0.007)

0.01 (0.007)

0.69 (0.109) t 2.46 (0.271) 0.79 (0.148)

0.41 (0.155)

3.24 (0.350) 0.03 (0.002) 0.02 (0.004) t

1.07 0.03 0.02 0.02

0.02 (0.001)

0.01 (0.008) 0.01 (0.006)

0.03 (0.005) 0.38 (0.038) 0.27 (0.029)

0.05 (0.018) 0.40 (0.174) 0.29 (0.040)

0.01 (0.008) 0.10 (0.013)

0.02 (0.001) 0.17 (0.007)

0.20 (0.020)

0.25 (0.019)

0.03 (0.002) t 0.01 (0.014) 0.02 (0.013)

0.75 (0.349) 0.40 (0.140) (0.487) (0.003) (0.002) (0.005)

0.03 (0.002) 0.04 (0.003) 0.02 (0.001) 0.02 (0.006)

(continued on next page)

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Table 1 (continued) RIb

Peak #

RIc

75 1500 1708 76 1499 1730 77 1507 1732 78 1509 1724 79 1526 1761 80 1657 2150 81 1670 2181 82 1677 2271 83 1687 2220 84 1697 2254 85 1754 2308 86 1808 2277 87 2297 2344 Monoterpene hydrocarbonse Oxygenated monoterpenese Sesquiterpenese Otherse

Compound

‘Femminello’ lemon

Bicyclogermacrene cis-α-Bisabolene E,E-α-Farnesene β-Bisabolene δ-Cadinene trans-β-Santalol epi-Santalene cis-Nerolidol acetate α-Bisabolold β-Sinensal α-Sinensal Nootkatone Tricosane

0.02 (0.001)

‘Valencia’ orange

0.02 (0.001) 0.40 (0.008) 0.01 (0.000)

V + F hybrid % (± SD) 0.02 0.02 0.21 0.01 0.01

(0.001) (0.001) (0.008) (0.001) (0.001)

0.02 0.02 0.09 0.02

(0.001) (0.001) (0.004) (0.002)

0.03 (0.001) 0.05 (0.001)

0.02 (0.001) 0.03 (0.002) 76.43A 20.37E 1.14E 1.07D

0.05 (0.008) 0.02 (0.001) 0.02 (0.002) 94.16C 3.22B 0.20A 2.31E

2n cybrid

4n cybrid

0.02 (0.002) 0.02 (0.002)

0.04 (0.006) 0.02 (0.002)

0.31 (0.031)

0.39 (0.032)

0.01 (0.005)

0.02 (0.003)

0.03 (0.004)

0.04 (0.007)

0.01 (0.008) 97.27D 1.78A 0.67B 0.22A

88.66B 9.93D 0.74C 0.56C

93.98C 4.58C 0.98D 0.31B

a Values (relative peak area percent) represent averages of 18 determinations for each cybrid and hybrid (nine for each collection year: 2006 and 2007), and 9 determinations for parents (collection year 2007), (t = trace, b 0.01%); b Retention index (KI) relative to standard mixture of n-alkanes on SPB-5 column. c Retention index (KI) relative to standard mixture of n-alkanes on Innovax column. d Co-elution with authentic standard. e Different letters in the same line represent significant difference at p ≤ 0.01 by HSD Tukey's test.

showed a strongly inhibition of some components (i.e. sesquiterpene hydrocarbons), as well as an overproduction of the other ones (i.e. citronellal); these contrasting results prompted some authors to claim that Citrus somatic hybrids do not retain their parental traits (Gancel et al., 2005a). In the present study we have instead observed a slight overproduction of some monoterpene hydrocarbons (see Fig. 2) limited only to the two cybrids, and a widespread reduction of several oxygenated components with respect to one or both parents (Table 1). The results of this study show, analogously to many similar ones, just how difficult it is to establish an inheritance mechanism related to the biosynthetic accumulation of secondary metabolites throughout various breeding methodologies of Citrus species (Fabroni, Ruberto, & Rapisarda, 2012; Rapisarda et al., 2003; Ruberto & Rapisarda, 2002; Ruberto, Renda, Piattelli, Rapisarda, & Starrantino, 1997b; Ruberto, Starrantino, & Rapisarda, 1999; Ruberto et al., 1997a). This is probably due to the complexity and genetic changeability of this genus (Barkley, Roose, Krueger, & Federici, 2006; Guo, Cheng, Chen, & Deng, 2006). Comparison between the essential oils suggested that both parents may have contributed to the composition of the hybrid and cybrids here examined. The hybrid and cybrids exhibited, in fact,

inherited traits from both of their parents in different proportions. These comparisons clearly provide an insight into the spectrum of changes associated with the genetic manipulation by protoplast fusion. It is important to couple such observations with detailed analyses of chromosomal events occurring in citrus, including ploidy changes and the extent of genomic asymmetry and somatic recombination. Chromosome-painting techniques could play a crucial part in achieving these objectives. Genomic in situ hybridization (GISH) is an efficient and accurate technique able to discriminate chromosomes between different plant species in their hybrids (Mukai & Gill, 1991). In Citrus, GISH has been successfully applied for cytological characterization of intergeneric somatic hybrids (Fu, Chen, Guo, & Deng, 2004). Our goal was to determine the presence of ‘Valencia’ chromosomes and the occurrence of any translocations between genomes in ‘Valencia+ Femminello’ cybrids. The presence of the GISH signal proved the contribution of ‘Valencia’ chromosomes in 2n and 4n cybrids (Fatta del Bosco, Tusa, & Galasso, 2003), confirming that chromosomal rearrangement occurred during the fusion and regeneration processes of our cybrids. Small introgression from ‘Valencia’ sweet

7 100 6

90 80

5

70

[%]

[%]

60 50

4 3

40 2

30 20

1

10 0

0 2nCybrid

4nCybrid

β -Pinene

V+Fhybrid

Limonene

'Femminello' Lemon

'Valencia' Orange

γ-Terpinene

Fig. 2. Content of β-pinene, limonene and γ-terpinene in 2n and 4n cybrids, V+ F hybrid and in both parents.

2nCybrid

Nerol

4nCybrid

Neral

V+Fhybrid

'Femminello' Lemon

Geraniol

'Valencia' Orange

Geranial

Fig. 3. Content of nerol, neral, geraniol and geranial in the 2n and 4n cybrids, V + F hybrid, and in both parents.

L. Abbate et al. / Food Research International 48 (2012) 284–290

289

Acireale CT) for helpful assistance in statistical analyses, and Mr. Vincenzo Marino (IGV-CNR, Palermo) for skillful technical assistance.

References

Fig. 4. Discriminant score plot (functions 1 and 2) of all components of the essential oils.

orange might be at the origin of the new traits shown in both lemon cybrids.

4. Conclusions Inter-specific somatic hybrid and cybrid plants between C. sinensis and C. limon have been obtained in an effort to combine qualitative specific characters from both parental genotypes. The study showed that citrus somatic hybrids may produce an essential oil whose chemical composition exhibits original features with respect to those of both parents. The presence of distinctive traits in the essential oil profiles is a further demonstration of the high potential of somatic hybridization or cybridization in citrus cultivar improvement. In particular, concerning the new varieties here described it is actually very difficult to establish if they will experience a future production. They, as showed in Fig. 1, are quite different: the somatic hybrid V + F according to colour and shape is very similar to an orange, whereas the two cybrids, for the same reasons, can be considered two kinds of lemon fruits. From a commercial point of view both cybrids could be two new successful fruits, being endowed with an original and pleasant sensorial profile. However, only the consumer's acceptance and the processing industry could confer their future and desirable success.

Acknowledgement This work was financially supported by MIUR Project: “Risorse genetiche vegetali per la produzione di sostanze di interesse per la salute umana”. The authors wish to thank Mr. Antonio Giuffrida (CRA-ACM,

Table 2 Standardised determinant function coefficient of selected components. Compound

Function 1

Function 2

α-Thujene α-Pinene β-Pinene Myrcene p-Mentha-1(7),8-diene Limonene trans-β-Ocimene γ-Terpinene α-Terpinolene Linalool cis-p-Mentha-2,8-dien-1-ol cis-Chrysanthenol β-Sinensal

− 7.658 19.072 − 16.932 − 3.386 4.276 5.439 1.098 7.352 − 4.797 5.841 − 6.042 − 1.255 − 0.295

− 5.170 6.853 1.128 1.024 1.752 − 3.046 2.737 0.704 0.775 1.020 3.941 2.054 − 3.142

Functions 1 and 2 from the linear discriminant analysis (LDA).

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