Somatic and Sexual Hybrids
The first example here reported regarding the study of the chemical profiles of new Citrus hybrids concerns the hybrids from the crossbreeding of the “Valencia” sweet orange (Citrus sinensis L. Osbeck) + the “Femminello” lemon (Citrus limon L. Burm) to obtain the allotetraploid somatic hybrid (Valencia + Femminello), which in turn by back crosses with
“Femminello” lemon allowed to obtain three sexual hybrids, named Fx(V + F)5, Fx(V + F)7 and Fx(V + F)10.
The somatic hybrid V + F is quite similar in shape, size and colour to that of “Valencia”
sweet orange parent, whereas fruit aspect of the three selected sexual hybrids, Fx(V + F)5, Fx(V + F)7 and Fx(V + F)10, coming from interploid (4n x 2n) backcross with “Femminello”
lemon was similar to that of the C. limon maternal parent, with the partial exception of Fx(V + F)7, which shows a color and a size more similar to the V + F parent [155].
The polyphenol profile of the juices of all the aforesaid samples, namely parents and hybrids, has been studied by a combination of liquid-chromatography-ultraviolet-diode-array- detector-mass-spectrometry (LC-UV-DAD-MS). Ten flavonoids (TF), comprising seven flavanones and three flavones, and four hydroxycinnamic acids (HCA) have been characterized and quantified in all hybrids and compared with those of respective parents. The
“Valencia” + “Femminello” somatic hybrid shows an intermediate polyphenol composition with respect to those of parents, with a slight prevalence of lemon influence. The three sexual hybrids show, instead, different and more complex chromatographic profiles.
The same aforesaid Citrus samples have been subjected to a further phytochemical study concerning the analysis of their peel essential oils [44]. The oils were obtained by hydrodistillation of the flavedo portion of the peel of each variety, and their composition has been studied by gas chromatography (GC) combined with a flame ionization detector (FID) and a mass spectrometry (MS). In total, 83 components were fully characterized and grouped in four classes (monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpenes, and others) for an easier comparison of all oils. The volatile profile of all hybrids is strongly influenced by that of “Femminello” lemon parent, however, a different behavior in the peel essential oil composition of the allotetraploid hybrid and the three hybrids is observed.
POLYPHENOL PROFILE. Table 1 lists the quantitative profile of the ten flavonoids (TF) and four hydroxycinnamic acids (HCA) reported in Figure 5, individuated in “Valencia”
orange, “Femminello” lemon and their hybrids.
The profile of “Valencia” sweet orange (V) is characterised by the predominance of two flavanones, hesperidin and narirutin, which together constitute 93% of this subclass, whereas vicenin-2 is the main and almost unique flavone. Amongst HCA, the ferulic is the main one, sinapic and coumaric follow at after a considerable gap, caffeic is found at trace level.
“Femminello” lemon shows a greater amount of TF than “Valencia” orange, hesperidin and eriocitrin being the most important flavanones (97% of total subclass); diosmetin 6,8-di-C- glucoside is the main flavone, followed by vicenin-2 and diosmin. Concerning HCA, also in this case ferulic is the main acid, unlike “Valencia” orange, sinapic and coumaric are present in higher amounts, caffeic is still at trace level. The composition of both parents does not present significant differences with the average composition of this species as reported in literature [159-164].
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Figure 5. Polyphenols from Citrus juices.
Table 1. Polyphenols (mg/L) in juices of “Valencia” orange, “Femminello” lemon and their hybrids
# Components Valencia
(V)
Femminello (F)
Valencia + Femminello (V + F)
F x (V + F)5 F x (V + F)7 F x (V + F)10
Total Flavonoids 135.64 346.42 243.13 167.33 267.90 381.18
Flavanones 108.04 199.84 182.17 79.80 235.26 261.79
3 Eriocitrin 0.65 (0.08)a 70.74 (2.53)d 54.22 (0.05)c 43.45 (0.68)b 82.46 (0.63)e 112.12 (1.39)f
4 Narirutin 19.39 (1.10)e 0.96 (0.26)a 17.48 (0.27)d 0.57 (0.33)a 9.12 (0.17)c 3.20 (0.27)b
5 Naringenin 7-O-glc 1.04 (0.31)bc 2.69 (0.27)d 1.52 (0.35)c 0.38 (0.13)a 0.99 (0.06)bc 0.64 (0.13)ab
6 Naringin 1.82 (0.20)b 0.08 (0.04)a 1.30 (0.47)b 0.02 (0.01)a 0.13 (0.01)a 0.12 (0.09)a
7 Hesperidin 81.50 (0.22)b 124.58 (25.91)c 107.35 (0.43)bc 35.09 (0.48)a 141.20 (0.31)c 144.89 (7.16)c 9 Neohesperidin 0.64 (0.46)n.s 0.73 (0.20)n.s. 0.41 0.27)n.s. 0.27 (0.01)n.s. 0.76 (0.02)n.s. 0.75 (0.02)n.s.
10 Dydimin 2.99 (0.00)d 0.06 (0.01)ab 0.10 (0.00)b 0.03 (0.00)a 0.59 (0.02)c 0.07 (0.05)ab
Flavones 27.28 145.93 60.74 87.08 32.01 118.37
1 Vicenin-2 26.50 (2.17)d 13.46 (0.10)b 25.21 (1.68)d 10.27 (0.03)ab 8.54 (1.60)a 19.13 ((0.06)c 2 Diosmetin 6,8-di-C-glc 0.41 (0.17)a 109.82 (0.39)f 35.21 (1.25)c 48.53 (0.02)d 17.19 (0.39)b 57.89 (0.17)e 8 Diosmin 0.37 (0.30)a 22.64 (19.60)abc 0.31 (0.16)a 28.27 (0.15)bc 6.28 (0.30)ab 41.35 (8.48)c
Hydroxycinnamic Acids 16.50 11.45 16.38 10.21 17.35 8.44
1* Caffeic acid 0.78 (0.00)d 0.27 (0.01)a 0.68 (0.00)c 0.46 (0.04)b 1.05 (0.00)e 0.30 (0.01)a 2* Coumaric acid 1.17 (0.00)a 2.33 (0.03)c 0.98 (0.00)a 1.90 ((0.31)b 1.97 (0.05)b 0.97 (0.13)a 3* Ferulic acid 13.05 (0.04)e 6.59 (0.04)c 11.72 (0.04)d 5.31 (0.42)b 12.94 (0.01)e 3.84 (0.07)a 4* Sinapic acid 1.51 (0.02)ab 2.26 (0.08)c 1.74 (0.02)b 2.58 (0.22)d 1.39 (0.00)a 2.67 (0.04)d Values represent averages of three determinations and standard deviation (± SD) is given in parentheses; different letter in the same row represents
significant difference at p ≤ 0.01 by Duncan‟s multiple range test; n.s. = non significant.
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Figure 6. Content (mg/L) of total flavonoids (T.F.), comprising flavanones and flavones, and hydroxycinnamic acids (H.A.), in the juices of “Valencia” orange (V), “Femminello” lemon (F) and their hybrids: V + F, Fx(V + F)5, Fx(V + F)7 and Fx(V + F)10.
The compositional polyphenol profile of the allotetraploid somatic hybrid V + F is intermediate between those of parents, with a slight prevalence of lemon influence.
Hesperidin and eriocitrin are the main flavanones as in lemon, analogously diosmetin 6,8-C- glucoside, as in lemon, is the most important flavone. Concerning the HCA, a closer behaviour to “Valencia” orange is instead observed.
The sexual hybrids show a variegated profile. In fact, the hybrid Fx(V + F)5 is the sole sample of this group showing a slight predominance of flavones with respect to flavanones, and unlike both parents eriocitrin is predominant with respect to hesperidin. The hybrids Fx(V + F)7 and Fx(V + F)10 show a higher level of flavanones than both parents, with the usual predominance of hesperidin over eriocitrin; however, unlike the previous hybrid, a higher amount of naritutin is observed. Instead the flavones content is quite different: Fx(V + F)7 has a lower concentration of these components than both parents, whereas Fx(V + F)10 has an intermediate amount, with the exception of diosmin, whose amount is decidedly higher than both parents. The HCA amount of Fx(V + F)5 and Fx(V + F)10 are comparable to lemon, whereas those of Fx(V + F)7 are much more similar to those of V + F. The graph of Figure 6 gives an immediate comparison of the content of the previous described components in orange, lemon and their hybrids.
In order to refine these quantitative data with the aim to obtain an optimal differentiation of all species here involved, namely parents and hybrids, a multivariate analysis using the quantitative data of total flavonoids (TF) and hydroxycinnamic acids (HA) has been applied.
The discriminant analysis afforded the separation shown in Figure 7. The graphic representation in the two functions: namely, functions 1 (94.1%) and 2 (3.7%) representing 97.8% of the total variance, shows the large differentiation between “Valencia” orange and
“Femminello” lemon. The somatic hybrid V + F is placed between both parents according to
Citrus Genetic Improvement 153 the two functions, being closer to the Femminello parent in both cases. The three sexual hybrids, Fx(V + F)5, Fx(V + F)7 and Fx(V + F)10, show a more irregular behaviour, confirming the previous considerations. It should be underlined that in this case their parents, namely F and V + F, are closer than the previous two parents V and F, in particular for the first and more significant function.
Figure 7. Discriminant score plot (Functions 1 and 2) of polyphenols (TF and HA) in the juices of
“Femminello” lemon, “Valencia” orange, and their hybrids V + F, Fx(V + F)5, Fx(V + F)7 and Fx(V + F)10.
ESSENTIAL OIL PROFILE. The chemical composition of the essential oils of the hybrids and their parents, obtained by the peel hydrodistillation, have been studied by gas chromatography (GC) combined with a flame ionization detector (FID) and a mass spectrometry (MS), and Table 2 lists the composition of the essential oils of all samples. The 83 identified components were grouped into four classes: monoterpene hydrocarbons (15 components), oxygenated monoterpenes (31 compounds), sesquiterpenes (20 compounds) and others (17 compounds). 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.