Industrial Crops and Products 32 (2010) 639649

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Industrial Crops and Products

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Essential oils, genetic relationships and in vitro establishment of Helichrysum

italicum (Roth) G. Don ssp. italicum from wild Mediterranean germplasm
I. Morone-Fortunato a , C. Montemurro b , C. Ruta a, , R. Perrini a , W. Sabetta b , A. Blanco b ,
E. Lorusso c , P. Avato c
a
Dipartimento di Scienze delle Produzioni vegetali, Universit Aldo Moro, Via Amendola 165/A, I-70125 Bari, Italy
b
Dipartimento di Biologia e Chimica Agro-Forestale ed Ambientale, sezione di Genetica, Universit Aldo, Moro, Via Amendola 165/A, I-70125 Bari, Italy
c
Dipartimento Farmaco-Chimico, Universit Aldo Moro, Via Orabona 4, I-70125 Bari, Italy

a r t i c l e i n f o a b s t r a c t

Article history: In this paper we describe the morphological, histological and chemical characters of 20 native Helichry-
Received 6 May 2010 sum italicum (Roth) G. Don ssp. italicum genotypes collected from different locations in Italy and Corsica
Received in revised form 26 July 2010 (France) and grown under similar edaphic and climatic conditions. An AFLP technique was used to exam-
Accepted 30 July 2010
ine the level of genetic variability among the genotypes with the aim to disclose a possible correlation
between the genetic and chemical data. The chemical analysis recognizes at least three different chemo-
types based on the major constituents in the essential oils. This was conrmed by the AFLP analysis
Keywords:
resulting in a dendrogram divided into three main clades. Because of the polymorphic trait of Helichry-
Asteraceae
Helichrysum italicum ssp. italicum
sum species, an efcient micropropagation protocol was established for the 20 H. italicum (Roth) G. Don
Essential oil ssp. italicum genotypes to guarantee the availability of stable genetic material with dened chemical
Secretory glands proles for industrial applications. The genotypes were found to inuence the in vitro performance and
AFLP markers the chemical composition of the essential oils, but not the phenotypic traits of the plants.
Micropropagation 2010 Elsevier B.V. All rights reserved.

1. Introduction
The genus Helichrysum (Miller) belongs to the Asteraceae fam-
ily and is a very large genus including approximately 600 species
widespread all over the world. The genus is represented in the
Mediterranean area by nearly 25 native species. Among them, eight
species are present in Italy: Helichrysum italicum (Roth) G. Don; H.
frigidum (Labill.) Willd. (endemic of Sardinia and Corsica); H. mon-
telinasanum E. Schmid and H. saxatile Moris (restricted to Sardinia,
Monte Linas); H. nebrodense Heldr. and H. siculum (Sprengel) Boiss.
(endemic of Sicily); H. rupestre (Ran.) DC.; H. stoechas (Pignatti,
1984). The strong trend of Helichrysum species to show a high
level of anatomical and morphological polymorphism (Jahn and
Schnfelder, 1995), creates a diversity of several varieties and/or
ecotypes (Pignatti, 1984). A high chemical and genetic diversity
has also been reported (Harborne and Turner, 1984; Angioni et al.,
2003; Tundis et al., 2005). H. italicum (syn. H. angustifolium DC) is the
most widespread species in the Italian environment and includes
two subspecies, H. italicum ssp. italicum and H. italicum ssp. micro-
phyllum (Willd.) Nyman (Pignatti, 1984). Besides its ornamental
value, the success of this species is also due to the several activ-
Corresponding author. Tel.: +39 0805443045; fax: +39 0805442976.
E-mail address: c.ruta@agr.uniba.it (C. Ruta).
ities related to the essential oils produced by the glandular hairs
present on the leaves and ower heads of the plant.
Metabolites isolated from H. italicum and especially its volatile
oil have been found to display many biological properties, such as
the anti-inammatory (Sala et al., 2001, 2002, 2003; Appendino
et al., 2007; Voinchet and Giraud-Robert, 2007), anti-allergic and
antimicrobial (Chinou et al., 1997; Nostro et al., 2001, 2002, 2004;
Voinchet and Giraud-Robert, 2007), antioxidant (Sala et al., 2002)
and anti-viral (Nostro et al., 2003; Appendino et al., 2007). Even
though this large genus has been extensively investigated, the
chemical variations and the genetic relationships within the same
species still remain unclear. This is especially true for the essential
oil composition and the genetic relationship of genotypes belong-
ing to H. italicum (Roth) G. Don species. Compared to other plants,
the use of molecular markers to exploit its genetic variability has
also been limited. Angioni et al. (2003) carried out a molecular
characterization of two H. italicum chemotypes with RAPD (ran-
dom amplied polymorphism DNA) technique and Smissen et al.
(2007) used AFLP (amplied fragment length polymorphism) n-
gerprinting to investigate intergeneric hybridization between the
endemic New Zealand species of H. lanceolatum and Anaphalioides
bellidioides.
Following our previous investigation on H. italicum ssp. micro-
phyllum (Perrini et al., 2009b), we describe the morphological,
histological and chemical characters of 20 wild H. italicum (Roth)

0926-6690/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.indcrop.2010.07.023
640 I. Morone-Fortunato et al. / Industrial Crops and Products 32 (2010) 639649
Table 1
Genotypes collection and relative code number of H. italicum (Roth) G. Don ssp.
italicum.
Code number Genotype
2 Brindisi di Montagna (Potenza)
3 Localit Centopozzi (Foggia)
5 Localit Baia di Crovani (Corsica)
6 Marina di Pulsano (Taranto)
7 Monopoli (Bari)
8 Noci (Bari)
9 Orto Botanico (Bari)
10 Policoro (Matera)
11 Localit Pollino, Sanseverino Lucano (Potenza)
13 Putignano (Bari)
14 Ruggiano, Manfredonia (Foggia)
15 Sambuchello (Foggia)
16 Sammichele di Bari (Bari)
17 Localit Torre Gattarella, Vieste (Foggia)
18 Localit Valle del Sinni (Basilicata)
19 Localit Valle dellInferno (Foggia)
20 Locorotondo (Bari)
23 Localit Punta Polveraia (Isola DElba)
26 Orto Botanico (Siena)
27 Termini Imerese (Palermo)
G. Don ssp. italicum genotypes collected from different locations
in Italy and from Corsica (France) and then grown under the same
edaphic and climatic conditions at Bari, Italy. Moreover, AFLP mark-
ers were used to examine the level of genetic variability among the
20 genotypes with the objective to disclose a possible correlation
between genetic and chemical data.
The polymorphic traits of Helichrysum species cannot guarantee
stable genetic material with dened chemical proles for industrial
applications. To overcome this, an efcient micropropagation pro-
tocol to reproduce and stabilize selected clones of H. italicum ssp.
michrophyllum was recently established by our group (Perrini et al.,
2009b). The usefulness of this protocol was veried in the current
study on H. italicum ssp. italicum genotypes.
2. Materials and methods
2.1. Plant material
Twenty H. italicum (Roth) G. Don ssp. italicum (Table 1 and
Fig. 1) genotypes collected from different areas of Italy and
Corsica (France) were maintained in an open-eld (La Pietra
farmMonopoli countryside, Bari, South Italy) and irrigated when
necessary through micro-irrigation applying water at a volume
of 300 m3 /ha. The soil was characterized as red in colour, with a
medium depth, good structure and medium fertility (1.336 g kg1
N; 127.78 mg kg1 P2 O5 ; 268.23 mg kg1 K2 O; 2.08% organic mat-
ter; pH = 7.7). The climate of the area is typically Mediterranean.
During mild, rainy winters, the average monthly temperature is
around 9.5 C and during hot, dry summers 23.2 C. The mean
annual precipitation is 573 mm of which 36% from April to Septem-
ber with a relative humidity of 68%.
Genotypes are identied by a code number referring to the loca-
tion of collection of the plants (Table 1).
2.2. Taxonomic identication
The Italian National Flora was used for taxonomic identication
(Pignatti, 1984). The morphological parameters used were basal
leaves length, basal leaves width, trichomes diameter, trichomes
basal cell diameter, external bracts glandular hairs, number of ow-
ers/head, number of heads/corymb, and head diameters.
2.3. Histology
Closed ower section and sections (4050 m thick) obtained
by sliding microtome of fresh leaf tissues from plants grown in the
eld and in vitro propagated were stained with Sudan IV (Gomori,
1952). Single owers and leaf sections were mounted on slides
to examine the secretory glands directly under a light microscope
(Zeiss).
2.4. Analysis of the essential oils
Plants of the different H. italicum (Roth) G. Don ssp. italicum
genotypes were harvested at the owering stage and air-dried
before the extraction of the essential oils. Dried ower heads were
steam-distilled to recover the essential oils (2 h 30 min; with a
Spring type apparatus, Albrigi, Italy). Oils were kept in the refriger-
ator until analyzed.
A Trace GC Ultra Thermo Finnigan gas chromatograph equipped
with a FID detector was used for the compositional analysis of the
essential oils. Samples (1 l) were injected in the cold on-column
mode in a DB-5 (J&W Scientic) fused silica capillary column
of 30 m 0.25 mm; 0.25 m lm thickness. The chromatographic
conditions were as follows: detector temperature 300 C; column
temperature was programmed from 60 C (5 min isothermal) to
270 C (30 min isothermal) at 4 C/min. Arithmetic indices (AI)
were measured against a series of straight-chain alkanes (C7C27).
Hydrogen was the carrier gas (35 kP; 2.0 ml/min). Data were pro-
cessed using the Chrom-Card 32-bit computing software.
Gas chromatographymass spectrometry (GCMS) analyses
were performed with a Hewlett Packard 6890-5973 mass spec-
trometer interfaced with a HP Chemstation according to Avato et
al. (2005). Samples (1 l) were injected using the splitless sam-
pling technique. Identication of the oil constituents was based on
comparison of their GC retention times in combination with arith-
metic indices (AI) and by means of reference mass spectra from
standard compounds and/or from library les (Joulain and Knig,
1998; Avato et al., 2005; Adams, 2007).
2.5. Genetic analysis
DNA was extracted from 50 mg of fresh leaves with the Gene
Elute Plant kit (Sigma) following the manufactures instructions.
DNA concentration was determined using a spectrophotometer at
260 nm, and by means of electrophoresis on 0.8% agarose gel with
DNA standard.
AFLP analysis was conducted essentially as described by Vos
et al. (1995) with some modications according to the indica-
tions reported in Montemurro et al. (2005, 2008). Genomic DNA
(250 ng) was double digested for 1 h at 37 C in a nal volume of
40 l with 10 units of PstI and 10 units of MseI (New England Bio-
lab), and 1 R/L restriction/ligation buffer (10 mM TrisHCl, pH 7.5,
10 mM magnesium acetate, 50 mM potassium acetate). To this mix-
ture 10 l of a ligation mix containing 50 pmol of double-stranded
adapters for MseI and 5 pmol adapters for PstI, 3.5 units T4 ligase
(Invitrogen) and 1 R/L restriction/ligation buffer, were added and
let to react overnight at 15 C. Thirty microliters of the resulting
digestionligation mixture were used, without dilution, for PCR
pre-amplication by adding 1 PCR buffer Euroclone (160 mM
(NH4 )2 SO4 , 670 mM TrisHCl, pH 8.8, 0.1% Tween 20), 50 mM
MgCl2 , 75 ng of primer MseI (+1N) and 375 ng of primer PstI (+1N),
0.2 mM of each dNTP, 2 units of Taq DNA polymerase (Euroclone),
in a nal volume of 50 l. PCR thermal conditions were 21 cycles
of 30 s at 94 C, 1 min at 56 C, and 1 min at 72 C. A I-Cycler (Bio-
rad) thermal cycler was used. Five microliters of pre-amplication
products were used as a template for selective amplication by
adding 1 PCR buffer Euroclone (160 mM (NH4 )2 SO4 , 670 mM
 I. Morone-Fortunato et al. / Industrial Crops and Products 32 (2010) 639649
Fig. 1. Four selected genotypes (5-7-9-26) among the analyzed clones of H. italicum (Roth) G. Don ssp. italicum.
TrisHCl, pH 8.8, 0.1% Tween 20), 50 mM MgCl2 , 96 ng primer
MseI (5 -GATGAGTCCTGAGTAA-3 + 3N) and 3.2 ng primer PstI (5 -
AGACTGCGTACATGCAG-3 + 3N), 0.2 mM dNTP each, 0.5 units Taq
DNA polymerase (Euroclone), in a nal volume of 10 l. PstI primers
were radiolabelled with -[33P]-ATP. The following PCR condi-
tions were used: 94 C for 3 min, 12 cycles of 30 s at 94 C, 30 s
at 65 C (the annealing temperature was reduced every cycle by
0.7 C) and 1 min at 72 C. Twenty-three additional cycles were
then done at: 30 s at 94 C, 30 s at 56 C and 1 min at 72 C. Selec-
tive PCR was performed in a MJR-PTC200 thermal cycler. Three
primer combinations with three selective nucleotides were used.
Amplied fragments were separated using a denaturing 5% (w/v)
polyacrylamide gel electrophoresis (PAGE) (OWL Separation Sys-
tem), with a 100 bp DNA ladder (MBI-fermentas). Bands were
visualised by autoradiography and scored manually for their pres-
ence or absence.
AFLP polymorphic bands were scored as either present (1) or
absent (0) to create a binary matrix. All the molecular data were
analyzed using the NTSYS program. The SIMQUAL function, was
used to compute a similarity analysis of qualitative data by the Jac-
card index. The resulting matrix was subsequently analyzed by the
Unweighted Pair Group Method (UPGMA), and the similarity tree
was obtained with the SAHN clustering program.
2.6. Micropropagation
2.6.1. Establishment and multiplication stage
Apical and axillary buds, 45 mm length, were used as the
explant source. Buds were sterilized in a 0.1% (w/v) HgCl2 solu-
tion for 15 min and successively rinsed with distilled-sterile
water. For in vitro cultures, we used the basic culture medium
(BM), containing the macronutrients, the micronutrients, FeEDTA
(25 mg l1 ), thiamine HCl (0.4 mg l1 ), myoinositol (100 mg l1 ),
agar (7 g l1 ) according to Morone-Fortunato and Avato (2008).
The pH of the medium was adjusted to 5.65.8. Sterilization
of culture media was performed in an autoclave at 121 C for
641
20 min. Primary explants were cultured on BM enriched with
6-benzylaminopurine + indole-3-butyric acid (BAP 1 mg l1 + IBA
0.2 mg l1 ), with sucrose (20 g l1 ) added as the carbon source
(Perrini et al., 2009b). A total of 30 primary explants per geno-
type were put into 70 ml culture tubes (Sigma) containing 20 ml
medium. Microcuttings were established approximately after 4
weeks of culture, when shoots had reached 34 cm length and
developed 56 nodes. The efciency of the culture medium was
determined by recording the percentage of primary explants devel-
oping shoots, the number of shoots/explant and the shoot length.
Shoots were subcultured three times and then transferred onto
fresh multiplication media for proliferation.
2.6.2. Root formation stage
Each regenerated shoot was rooted in 500 ml jars containing
100 ml of BM with sucrose (30 g l1 ) but without growth regula-
tors or supplemented with indole-3-butyrrc acid (IBA 0.2 mg l1 )
(Perrini et al., 2009b). Each jar contained ve shoots; for each treat-
ment a total of 25 shoots were used. The percentage of rooting
shoots and the number of roots/shoot was scored after 15 days. All
cultures were incubated in a growth chamber at 21 1 C with a
photoperiod of 16 h light, under a light intensity of 50 E m2 s1 .
2.6.3. Acclimatization stage
The in vitro rooted young plants were rinsed with tap water
to remove adhering medium and individually transferred to the
greenhouse (constant 50% humidity level, and at 21 3 C temper-
ature), in Jiffy-Pots or in pots (8 cm diameter) with a peat mixture
(organic carbon 46%, organic nitrogen 12%, organic matter 80%)
mixed with perlite or sand at 1:1 (v/v) ratio. The percentage of
plants surviving was recorded after 30 days.
2.7. Cytology
Root tips were treated for 4 h with a 0.05% aqueous solution of
colchicine (Sigma) and then xed in ethanolacetic acid 3:1 (v/v).
642 I. Morone-Fortunato et al. / Industrial Crops and Products 32 (2010) 639649
Standard Feulgen-staining and squashing procedures were used to
obtain the preparations for the cytological analysis (Blanco et al.,
1996).
2.8. Statistical analysis
The experiments were conducted under a complete randomized
block design with 3 replications. Data were processed by analysis of
variance (ANOVA) and comparisons within and between the mean
values of treatments were made by the StudentNewmanKeuls
(SNK) test calculated at the condence level of P 0.01. Data
expressed as percentages were transformed using angular trans-
formation.
3. Results
3.1. Taxonomic identication
The morphological characters used in this study identied
all the genotypes as belonging to the H. italicum ssp. italicum.
Distinctive traits of each analyzed genotypes were in good agree-
ment with those reported in the literature (Table 2; Pignatti,
1984), except for the number of owers/head and the number of
heads/corymb. Observed differences for these characters reect the
distinct behaviour of the various genotypes even though they were
grown under the same conditions. Only genotypes 10, 11 and 14
have in fact the number of heads/corymb comparable to values
reported in the literature (Pignatti, 1984). Genotypes 2, 3, 23 and
27 developed the number of heads/corymb of 42, 48, 38 and 43,
respectively, that is slightly higher than reported, while genotypes
6, 8, 15, 16 and 26 showed much higher values, 80, 78, 72, 80, and
77, respectively (Table 2). The remaining genotypes showed inter-
mediate values in the range of 5861 heads/corymb. Another trait
worth noting is the low density or total absence of secretory glands
on the external bracts of the ower heads, while there is a very high
density of glands on each tubular ower (Fig. 2A).
3.2. Histology
Histological studies of the ower heads, and the fresh leaves of
H. italicum ssp. italicum indicated that glandular trichomes secret-
ing essential oils are present in both organs and are of one single
morphological type and for all genotypes formed by 12 cells (Fig. 2A
and B), divided into three zones (Table 3). These observations are
in agreement with those previously reported for H. italicum ssp.
microphyllum (Perrini et al., 2009b). The inspection of the secretory
glands of H. italicum ssp. italicum genotypes was generally easier
on the tubular corolla of single closed owers than on leaves due
to the presence of thick hairs on leaves. Therefore, the counting of
the gland number was not possible due to their very dense distri-
bution on the owers and to the presence of many non-glandular
trichomes on the leaves and stems. The diameter of non-glandular
trichomes and their basal cell were of 49 and 1720 m, respec-
tively (Table 2) agreeing with the literature (Pignatti, 1984).
3.3. Chemistry
The amount of essential oils recovered from the ower heads of
the different genotypes of H. italicum ssp. italicum under investiga-
tion was in general quite low, ranging from 0.1 to 0.2%. The highest
production of essential oils was found in the two genotypes 5 (0.4%)
and 16 (0.5%).
The chemical proling by means of GC and GC/MS analyses
allowed to group the genotypes according to the major constituents
present in their essential oils. Some of the genotypes (3, 5, 7 and 23)
produce an essential oils with a consistent or predominant amount
of monoterpenoids, in total ranging from 26.4% in genotype 23 to
53.6% in genotype 5, with nerol and its esters, neryl acetate and
neryl propanoate as the main constituents (Table 4).
The other genotypes are characterized by essential oils very rich
in sesquiterpenoids (from 61.6%, in genotype 15 to 91.3%, in geno-
type 20). Among these, essential oils of genotypes 2, 6, 8, 9, 13,
16, 18 and 20 can be distinguished by the high content of both
- and -selinene accounting from 35.8% (genotype 13) to 64.2%
(genotype 8) of the total. In contrast, some genotypes were char-
acterized by a high amount of either -selinene (10, 11 and 19) or
-selinene (14, 15), and in general those genotypes are peculiar in
that a decrease in the amount of selinene compounds is associated
with a relative increase in the content of -curcumene (Table 4).
Genotype 17 appears unique in the collection in that produces
32.3% of -bisabolene together with -curcumene (27.7%).
-Diketones represent another chemical class present to a
different extent in almost all genotypes, and were particu-
larly abundant in genotypes 3, 5 and 7 (12.9, 24.2, and 11.2%
respectively). Dominant -diketones are 4, 6, 9-trimethyldec-8-
en-3, 5-dione (5.511.0%) and 5, 7, 10-trimethylundec-9-en-4,
6-dione (2.66.9%). The -diketone 4, 6-dimethyloctan-3, 5-dione
(0.22.4%) appears instead as a common constituent in almost all
the oils (Table 4).
Among the minor monoterpene and sesquiterpene con-
stituents, carvacrol (1.614.8%), -caryophyllene (1.218.6%) and
ar-curcumene (1.18.3%) were found in discrete and variable
amounts in almost all the extracted essential oils (Table 4).
Finally, in genotypes 5 (2.2%), 8 (1.8%), 11 (3.8%), 15 (8.9%), 19
(7.9%), 23 (2.1%) and 27 (5.2%), rosifoliol represents a character-
istic component, not detected in the essential oils from the other
genotypes.
3.4. Genetic analysis
The AFLP analysis (Fig. 3) was conducted with three primer com-
binations and revealed a total of 357 amplied DNA fragments
ranging from 80 to 800 bp (Table 5). A total of 195 polymor-
phic bands were observed, but only 110 of these were considered
for the cluster analysis, excluding the highest and lowest molec-
ular weight bands that could create ambiguity in the analysis.
The polymorphism percentage ranged from 26% (P-AGG/M-ACG)
to 37% (P-AGC/M-ACT). Among the three tested primer combina-
tions, just one (P-AGG/M-ACG) was sufcient to distinguish the 20
genotypes.
The Diversity Index (DI) was calculated as: DI = 1 Pi2 , where Pi
is the phenotypic frequency for each assay unit (Russell et al., 1997).
This index expresses the ability of a marker system to discrimi-
nate between the samples used. It is equal to 0 for monomorphic
markers and approximate to 1 for a heterogeneous population. The
efciency of AFLPs in providing a large number of bands with just
one electrophoretic analysis is reected in the high value of the
mean of Diversity Index (92.1%), and in the number of separate
genotypes per assay unit (each AFLPs primer combination) that
ranged from 17 to 20.
Based on results from the 110 AFLP scored bands, a dendrogram
representing the genetic similarity of the 20 examined H. italicum
ssp. italicum genotypes was obtained (Fig. 4).
3.5. Micropropagation
In the current study, the micropropagation protocol used for
H. italicum ssp. microphyllum (Perrini et al., 2009b) was applied
to all the H. italicum ssp. italicum genotypes providing successful
regeneration.
 I. Morone-Fortunato et al. / Industrial Crops and Products 32 (2010) 639649 643

Table 2
Morphological parameters of H. italicum ssp. italicum genotypes plants compared with reference values.

Genotypes Basal leaves Basal leaves Heads/corymb (n) Flowers/heads (n) Heads diameter Trichomes Trichomes basal
width (mm) width (mm) (mm) diameter (m) cell diameter (m)

2 0.68 0.04 23.46 0.65 42.00 1.52 9.60 0.51 2.50 0.07 7.50 1.03 20.05 0.78
3 1.00 0.07 34.74 0.64 48.60 1.60 16.20 0.80 2.96 0.19 7.00 1.05 17.00 0.80
5 0.75 0.07 25.36 0.24 58.00 1.14 17.20 1.39 2.48 0.22 8.65 0.95 18.56 1.05
6 0.97 0.05 34.80 0.63 80.20 1.85 14.80 1.36 3.02 0.21 6.89 1.02 19.16 1.03
7 1.02 0.04 35.00 0.73 57.60 1.93 23.60 1.72 3.30 0.28 7.15 0.98 16.56 0.54
8 0.99 0.04 35.00 0.73 78.00 2.00 9.00 0.71 2.86 0.22 6.50 0.90 18.50 0.99
9 0.60 0.07 22.26 0.79 63.60 1.08 13.40 0.93 2.52 0.11 8.05 0.87 19.00 1.03
10 0.78 0.06 25.20 0.21 34.20 1.39 11.11 1.00 2.22 0.17 8.80 1.00 16.89 0.68
11 0.58 0.03 20.24 0.93 31.40 1.21 9.00 0.71 2.18 0.16 7.25 1.04 16.95 0.90
13 1.00 0.05 35.00 0.73 59.00 2.07 9.00 0.71 2.20 0.16 8.80 1.05 18.90 1.03
14 0.89 0.05 30.06 1.26 30.60 1.03 9.20 0.58 2.94 0.18 6.19 1.00 19.00 1.10
15 0.80 0.04 30.00 1.00 72.20 2.08 16.20 0.80 2.70 0.12 6.40 0.99 20.15 0.98
16 0.78 0.04 30.26 1.14 80.00 1.73 14.00 0.71 2.20 0.16 8.09 0.80 20.45 0.85
17 0.70 0.05 25.00 0.36 60.60 1.44 14.00 0.71 2.70 0.12 7.45 1.50 16.98 0.65
18 1.01 0.06 35.00 0.73 61.00 1.41 12.00 0.84 3.42 0.20 6.90 0.96 19.34 1.05
19 1.15 0.06 35.14 0.75 61.00 1.52 23.00 0.41 3.10 0.20 6.85 0.95 19.07 1.02
20 0.85 0.08 30.06 1.26 61.80 1.36 16.00 0.71 2.90 0.17 8.05 1.04 20.00 1.00
23 0.50 0.05 18.44 1.01 38.00 1.64 14.80 0.86 3.54 0.29 8.92 1.04 20.02 1.00
26 0.80 0.04 25.20 1.21 77.00 1.70 17.20 0.86 2.42 0.16 7.55 0.78 18.15 1.07
27 0.90 0.04 30.00 1.00 43.00 1.52 18.00 1.14 2.62 0.25 7.78 1.03 18.00 1.06

Reference values
Pignatti (1984) 0.51(1.5) 1535 2535 15 24,5 49 1720

Values are means of 10 repetitions SE.

Fig. 2. Glandular hairs of H. italicum ssp. italicum (A: tubular corolla of single closed ower 200; B: leaf 1000).

Table 3
Glandular trichomes of H. italicum ssp. italicum genotypes compared with reference values.

Genotypes Basal zone width (mm) Basal zone length (mm) Median zone diameter (m) Apical zone width (m) Apical zone length (m)

2 10.00 0.50 11.20 1.05 12.40 1.00 15.00 0.05 30.00 0.50
3 9.05 1.00 13.00 1.00 15.50 1.01 16.00 0.50 29.39 0.65
5 9.15 0.87 12.00 2.00 14.00 1.02 15.10 0.02 30.05 0.02
6 10.00 0.50 12.00 1.90 12.45 2.00 14.96 0.09 30.00 0.09
7 10.12 0.05 10.00 0.90 14.00 1.90 14.00 0.09 28.90 0.01
8 10.00 0.05 10.95 1.00 10.00 0.05 15.25 0.58 29.95 0.58
9 9.50 0.90 11.09 1.00 10.50 0.06 13.98 0.90 29.87 0.90
10 9.50 0.04 14.00 1.00 11.87 0.07 15.00 0.02 29.99 0.02
11 10.06 0.60 14.00 1.50 12.45 1.04 15.00 0.50 30.00 0.03
13 10.00 0.09 15.02 1.20 12.00 0.98 16.02 0.50 30.00 1.00
14 10.00 0.08 13.42 1.00 15.00 0.02 16.12 0.09 30.00 0.87
15 9.25 1.00 13.00 2.05 15.06 0.03 15.00 0.58 30.05 0.50
16 9.08 0.50 10.86 1.04 10.00 0.02 13.45 0.90 29.58 0.05
17 9.00 0.65 10.00 0.03 12.45 2.00 14.98 0.02 29.00 0.05
18 11.00 0.02 13.55 0.90 13.84 1.50 15.52 0.03 30.00 0.90
19 10.00 0.09 13.45 1.00 13.00 1.85 15.02 0.04 29.72 0.05
20 11.00 0.01 15.00 0.05 12.98 2.00 15.00 0.05 29.54 0.50
23 10.00 0.05 15.05 0.07 12.00 0.94 16.00 0.05 30.46 0.02
26 9.85 0.05 15.00 0.10 10.00 0.03 14.98 0.50 30.00 0.09
27 9.00 0.55 10.96 1.00 10.00 0.05 15.00 0.02 30.09 0.09

Reference values
Perrini et al. (2009) 10 1015 1015 15 30

Values are means of 10 repetitions SE.

 644
Table 4
Composition of the essential oils from H. italicum ssp. italicum genotypes.
a
Compounds AI % Identication

2 3 5 6 7 8 9 10 11 13 14 15 16 17 18 19 20 23 26 27 GC/MS

3,4-Hexanedione 800 6.8 11.3 2.6 GC/MS

Tricyclene 921 0.7 GC/MS
Artemisiatriene 923 2.1 GC, GC/MS
-Pinene 930 0.9 3.6 1.2 tr 0.8 0.5 1.1 GC, GC/MS
3-Octanone 979 0.7 2.8 GC, GC/MS
p-Cymene 1018 tr 1.2 0.5 0.5 1.7 tr tr 0.6 0.4 0.8 tr 0.5 0.5 1.8 tr 1.6 0.4 GC, GC/MS
1,8- Cineole 1024 3.3 4.1 0.3 GC, GC/MS
- Terpinene 1055 0.3 0.7 0.7 GC, GC/MS
2,4-Dimethyleptan-3,5-dione 1070 0.5 tr tr GC/MS
Linalool 1095 1.5 0.4 3.9 0.9 0.5 tr 1.0 1.4 0.4 tr 1.5 0.5 1.5 1.9 5.1 1.2 GC, GC/MS
Tagetone 1140 0.4 0.4 tr 0.9 0.3 0.4 0.4 0.4 0.4 GC, GC/MS

I. Morone-Fortunato et al. / Industrial Crops and Products 32 (2010) 639649

Terpinen-4-ol 1174 tr 0.2 1.8 2.2 1.2 4.2 GC, GC/MS
4,6-Dimethyloctan-3,5-dione 1180 0.9 0.8 2. 4 1.2 0.6 0.7 0.9 1.5 0.8 0.2 0.3 0.2 1.2 0.7 1.6 0.9 GC/MS
-Terpineol 1186 0.8 0.8 1.0 0.5 2.3 tr 1.5 1.0 0.8 0.9 0.5 2.6 0.4 1.0 0.9 2.9 1.2 0.4 0.6 GC, GC/MS
Nerol 1126 0.5 2.4 3.9 0.6 18.8 1.5 1.7 0.8 0.5 0.9 1.0 2.0 1.3 0.9 2.7 1.9 0.4 1.7 0.5 0.4 GC, GC/MS
Lavandulyl acetate 1290 0.7 0.7 0.9 0.4 1.0 0.2 tr 0.8 0.5 GC, GC/MS
Carvacrol 1300 3.8 6.2 1.9 8.4 9.8 6.7 6.8 14.8 8.7 3.8 1.6 9.5 7.4 9.8 5.4 2.6 5.8 3.3 3.2 3.6 GC, GC/MS
-Terpinylacetate 1346 0.4 tr GC, GC/MS
Neryl acetate 1360 1.7 15.1 32.0 0.4 8.5 0.7 3.2 1.0 1.1 0.8 1.8 4.5 1.4 1.0 2.8 4.7 1.4 11.4 8.1 1.2 GC, GC/MS
-Ylangene 1375 3.3 1.3 0.4 0.2 4.9 0.5 2.1 0.7 GC/MS
iso-Italicene 1403 0.4 tr 0.5 0.6 tr 0.7 1.2 0.4 GC/MS
Italicene 1407 0.7 2.7 0.9 1.4 1.7 0.8 1.3 0.4 1.1 1.9 0.6 2.5 0.7 1.9 1.5 6.5 1.6 GC/MS
-cis-Bergamotene 1415 2.6 0.6 0.7 1.0 1.3 GC/MS
-Caryophyllene 1420 5.1 1.2 3.0 2.7 tr 10.1 12.7 6.2 8.6 6.5 14.4 10.9 4.5 7.3 18.6 7.8 2.6 3.0 GC, GC/MS
-trans-Bergamotene 1430 2.9 0.3 1.1 1.1 0.5 1.4 GC/MS
4,6,9-Trimethyldec-8-en-3,5-dione 1438 5.5 11.0 6.2 tr GC/MS
-Humulene 1452 0.7 tr 1.2 0.9 0.7 0.7 0.3 0.6 0.7 0.3 0.7 0.6 3.2 1.7 0.8 GC, GC/MS
Neryl propanoate 1452 0.7 0.5 3.6 tr 6.8 2.3 2.7 0.2 tr 1.5 0.2 0.9 GC, GC/MS
-Acoradiene 1464 0.3 tr 6.7 0.2 0.2 GC/MS
-Acoradiene 1470 0.6 0.3 tr 0.2 GC/MS
4,5- di-epi-Aristolochene 1471 0.8 0.7 0.7 0.2 0.4 0.3 0.3 0.7 0.3 0.4 0.5 5.1 GC/MS
Selinen-4,11-diene 1478 8.1 6.0 7.4 4.8 3.3 3.1 4.2 7.1 2.9 5.1 1.2 6.9 3.3 6.0 1.4 2.0 GC/MS
-Curcumene 1479 4.2 23.3 5.0 2.3 6.5 3.3 8.4 1.6 15.0 14.3 3.4 27.7 17.1 14.7 7.7 41.0 14.5 GC/MS
ar-Curcumene 1480 4.9 6.4 8.3 6.4 2.9 1.1 3.1 2.7 6.1 2.2 GC/MS
-Amorphene 1483 3.2 4.0 GC/MS
2,4,6,9-Tetramethylundec-8-en-3,5-dione 1486 1.8 GC/MS
5,7,10-Trimethylundec-9-en-4,6-dione 1487 6.1 6.9 2.6 GC/MS
-Selinene 1489 33.3 3.6 19.7 38.0 25.7 15.9 11.6 20.0 2.8 3.8 24.5 2.3 28.9 15.3 26.7 6.1 22.0 4.7 GC/MS
-Selinene 1498 26.5 22.7 26.2 14.7 9.1 9.5 15.8 19.3 13.2 19.4 3.6 22.2 9.5 18.8 0.8 4.9 GC/MS
-Muurolene 1500 2.9 1.2 2.0 19.8 0.7 GC/MS
-Bisabolene 1505 1.1 32.3 GC/MS
7-epi--Selinene 1525 1.0 0.6 0.4 0.6 0.6 0.3 0.3 GC/MS
-Amorphene 1513 1.5 1.6 0.5 0.6 2.7 GC/MS
-Cadinene 1530 1.0 1.7 0.6 2.6 1.3 1.4 2.5 0.8 1.3 2.0 GC, GC/MS
-Cadinene 1540 0.3 0.2 0.2 0.4 0.3 GC/MS
-Calacorene 1544 0.7 0.4 0.8 GC/MS
Nerolidol 1561 1.3 0.3 0.3 0.2 1.5 GC, GC/MS
Geranyl butanoate 1562 0.5 2.0 GC, GC/MS
3,5,7,10-Tetramethylundec-9-en-4,6-dione 1570 0.5 3.4 GC/MS
Caryophyllene alchool 1573 0.6 0.4 1.1 GC/MS
-Caryophyllene oxide 1581 0.6 6.9 0.8 4.3 2.5 1.1 1.5 2.0 14.9 0.7 1.0 1.3 1.1 0.3 GC, GC/MS
Neryl isovalerate 1582 0.5 0.6 2.5 1.6 1.1 0.8 0.4 0.8 0.5 1.1 GC/MS
 I. Morone-Fortunato et al. / Industrial Crops and Products 32 (2010) 639649 645

Identication

GC, GC/MS

GC, GC/MS

GC, GC/MS
GC/MS

GC/MS
GC/MS
GC/MS
GC/MS

GC/MS
GC/MS
GC/MS
3.3
5.2

20.4
2.3
4.1

87.4
27

0.3

99.3
26

1.8
2.1

8.6
2.4
2.8

81.4
23

2.3

99.4
20

0.7
7.9

0.6
0.4
1.5
3.1

96.5
19

6.8

98.3
18

0.5

99.7
17

99.7
1.0
16

8.9

0.4
1.5
3.3

89.3
15

2.2

96.7
3.0
14

Fig. 3. AFLP electrophoretic patterns of 20 H. italicum genotypes (2-27) obtained

9.7
2.9

82.3
1.0

1.0
13

with the primer combination Pst-AGG/Mse-ACG.

2.1
3.8

11.1
1.6
2.2
4.7

97.6

3.5.1. Establishment and multiplication stage

11

The ANOVA analyses of percentage of proliferation, number of

shoots/explant, and growth showed signicant statistical differ-
1.1

13.7

6.1

98.9
2.0

10

ences among the investigated genotypes with the results reported

using the SNK test (Table 6). More than half of the H. italicum ssp.
Arithmetic indexes (AI) relative to a n-alkane series (C7 C27 ) calculated on a DB-5 column.
2.7

80.8

italicum genotypes responded to hormonal stimulation with prolif-

eration values greater than 50% (even 86100% for some genotypes)
(Table 6). Only four genotypes (7, 9, 11, and 13) showed a lower
0.7
1.8

1.1
0.6
7.0

98.0

proliferation percentage from 33 to 47% (Table 6).

The number of shoots/explant varied from a maximum value
0.4

1.8
3.0

99.0

of 6.53 (genotype 16) to a minimum of 1.47 (genotype 13). All

the neo-formed shoots always developed from axillary buds, never

from basal calli. The elongation of the shoots was also variable,
3.7

98.9

ranging from 0.77 to 2.87 cm (for genotypes 6 and 3, respectively),

with no relationship between the number of shoots and their
1.7
2.2

1.6
1.5

2.4
1.0

100

elongation.
5

98.7

3.5.2. Root formation stage

3

Analysis of variance (ANOVA) of the rooted shoots percentage

and number of new roots showed highly signicant genotypes
6.7

100

effect with the results of the SNK test presented in Table 7.

%

All the genotypes responded to rooting with values ranging from

50 to 100% of the shoots with roots. The only exception was geno-
1594
1606
1615
1620
1630
1648
1652
1668
1685
1727
AI

type 17, which naturally showed a lower rate of rooting (16.67%),

a

further decreasing with the addition of IBA (8.33%). Indeed, the cul-
7(11)-en-4-Eudesmol

ture medium enriched with IBA, increased rooting percentages in

Cubenol-1,10 di epi

Selin-11-en-4--ol

only three genotypes (2, 10, and 19).

Table 4 (Continued)

Guaiol acetate

The different response of the genotypes may be referred to three

-Eudesmol
-Eudesmol
-Eudesmol
Compounds

Neril tiglate

different conditions of endogenous hormones. The rst, covering

Rosifoliol

Yield %

the majority of our genotypes, characterized by an optimal hor-

Guaiol

monal balance, so that the addition of an exogenous hormone,

a

competitively, determines a decrease of rooting. In the second con-

646 I. Morone-Fortunato et al. / Industrial Crops and Products 32 (2010) 639649

Table 5
Characteristics of the banding patterns of the 3 AFLP primer combinations used among the 20 H. italicum ssp. italicum genotypes examined.

Primer combination Total bands (N) Polymorphic bands (N) Scored bands (N) Polymorphism (%) Genotypes per assay unit (N) Diversity index (%)

Pst-AGG/Mse-ACG 143 70 37 26 20 96.3

Pst-AGC/Mse-AGT 120 70 38 32 17 88.2
Pst-AGC/Mse-ACT 94 55 35 37 19 95.2

dition, involving only three genotypes, an increase of the hormone
concentration increases the percentage of rooting. Finally, the last
ve genotypes, which do not respond to exogenous hormones pos-
sibly have an optimal quantity of endogenous hormones, so that
they show 100% rooting and the further supply of growth regulators
does not inuence the endogenous hormonal balance determining
a decrease of rooting.
The number of roots obtained, although variable, allowed the
growth of plantlets suitable for acclimatization stage of H. italicum
ssp. italicum.

Table 6 3.5.3. Acclimatization stage

Proliferation, shoot number and shoot length of H. italicum ssp. italicum genotypes, The response to acclimatization of the 20 genotypes under study
after 30 days of in vitro culture. is described in Fig. 5. Plants growth was always satisfactory show-
Genotypes Proliferationa (%) Shoots/explantb (n) Shoot lengthb (cm) ing very high percentages (80% and 100%) of plants acclimatized
regardless of the use of Jiffy-Pots or conventional pots.
2 93.35ab 5.67ab 2.54ab
3 73.30abcde 2.93cdef 2.87a
5 53.34defg 4.67abed 2.50abc 3.6. Cytology
6 66.67bcdef 2.80cdef 0.77i
7 40.00fg 1.47f 1.27efghi
Caryological analysis conrmed that micropropagated clones of
8 93.33ab 3.27cdef 1.97bed
9 46.66efg 1.87ef 1.69cdefg H. italicum ssp. italicum had a chromosome number of 2n = 28, the
10 60.00cdefg 1.93ef 1.69cdefg same as the mother plant, agreeing with a report by Bacchetta et
11 33.30g 1.73ef 0.94ghi al. (2003) for the genus Helichrysum.
13 33.35g 1.47f 0.95ghi
14 86.67abc 4.20bede 1.50defgh
15 100.00a 5.13abc 1.90bede 4. Discussion
16 100.00a 6.53a 1.97bed
17 66.67bcdef 2.60def 1.20fghi The histological analysis showed the presence of glandular tri-
18 100.00a 3.00cdef 1.90bede
chomes on owers, leaves and young stems. The glands of all
19 80.00abed 2.80cdef 2.00bed
20 53.34defg 2.47def 1.44defghi H. italicum ssp. italicum genotypes were consistent with those
23 53.34defg 1.80ef 0.88hi reported by Perrini et al. (2009b) for the H. italicum ssp. micro-
26 100.00a 3.40cdef 0.87hi phyllum, both in structure and size, but, they differ from the at
27 100.00a 3.27cdef 1.75cdef glandular hairs (glands) of Arctium lappa, otherwise considered
Culture medium: BM + BAP 1 mg l1 + IBA 0.2 mg l1 + sucrose 20 g l1 . representative of the Asteraceae family (Fahn, 1979). Observations
Means followed by the same letters are not signicantly different at the P 0.01 made on leaves sections of in vitro propagated plants during the
level (SNK test).
a
multiplication stage, showed the presence of glandular hairs mor-
Percentage (%) is mean of 3 experiments. Each experiment comprises 30 primary
explants per genotype.
phologically identical to those observed on fresh plant material
b
Values are means of 50 repetitions. Each explant was a repetition. from the eld. Density of the glandular hairs is very high on the

Fig. 4. UPGMA dendrogram of 20 H. italicum genotypes obtained with data coming from three AFLP primer combinations. Brackets on the right indicate the two main clusters,
while letters a and b are referred to sub-groups of cluster 1.
 I. Morone-Fortunato et al. / Industrial Crops and Products 32 (2010) 639649 647

Table 7
Root formation and root number of H. italicum ssp. italicum genotypes, after 15 days of in vitro culture.

Genotypes Root formation (%) Roots (n)

1
Hormone free IBA (0.2 mg l ) Hormone free IBA (0.2 mg l1 )

2 66.67bc 91.67a 6.00bc 5.82bc

3 100.00a 91.68a 8.92bc 8.82bc
5 100.00a 91.69a 11.17bc 16.00b
6 100.00a 83.33ab 8.33bc 14.90b
7 100.00a 25.00e 7.25bc 3.67bc
8 100.00a 83.33ab 10.75bc 13.00bc
9 100.00a 91.67a 10.50bc 10.90bc
10 83.33ab 91.68a 8.80bc 9.36bc
11 100.00a 100.00a 9.42bc 12.67bc
13 100.00a 100.00a 8.00bc 13.00bc
14 100.00a 100.00a 10.25bc 13.00bc
15 58.33cd 41.67d 5.71bc 7.00bc
16 100.00a 50.00a 30.42a 26.83a
17 16.67ef 8.33f 2.00bc 4.00bc
18 66.67bc 50.00cd 5.25bc 3.33bc
19 83.33ab 100.00a 7.90bc 8.75bc
20 100.00a 100.00a 7.17bc 7.50bc
23 50,00cd 41.67d 1.60c 4.00bc
26 91.67a 83.33ab 8.64bc 10.80bc
27 100.00a 100.00a 12.17bc 12.42bc

Values are means of 50 repetitions. Each explant was a repetition. Means followed by the same letters are not signicantly different at the P 0.01 level (SNK test).

tubular corolla of each ower, while their density decreases very
much on the bracts covering the capitula, in contrast to H. italicum
ssp. microphyllum where secretory glands were observed with a
more or less constant density on all plant organs (Perrini et al.,
2009b).
Essential oils produced by the secretory glands of different
Helichrysum species have been studied and the analysis of H.
italicum ssp. italicum showed essentially three different chemo-
types for the essential oils (Bianchini et al., 2001; Paolini et al.,
2006; Satta et al., 1999; Tucker et al., 1997). Essential oils from H.
italicum ssp. italicum characterized by high amounts of monoter-
penoid constituents such as neryl acetate, neryl propanoate and
-pinene have been described by Bianchini et al. (2001) and Paolini
et al. (2006). A typical oil rich in geraniol (36%) and geranyl acetate
(15%) has been reported for plants collected in Greece (Bianchini et
al., 2001; Chinou et al., 1996). In contrast, other investigations rec-
ognized plant populations of H. italicum ssp. italicum with dominant
amounts of sesquiterpenes (Bianchini et al., 2001).
Despite the fact that fresh or dry ower heads of H. italicum are
included in several European Pharmacopoeias for ofcial use, to the
best of our knowledge, previous chemical studies on the essential
oils from this species have been substantially carried out on the
plant aerial parts, including owers, leaves and stems (Charles and
Simon, 1991; Bianchini et al., 2001; Paolini et al., 2006; Satta et
al., 1999; Tucker et al., 1997). Data reported here are the chemi-
cal characterization of the essential oils produced only by the plant
owers, which are especially rich in glandular structures as con-
rmed by our histological observations (Fig. 2). This work follows
our recent investigation (Perrini et al., 2009b) for the chemical char-
acterization of the essential oil from ower heads of H. italicum ssp.
microphyllum.
According to the results reported in Table 4, genotypes of H.
italicum ssp. italicum from different locations in Italy represent dif-
ferent chemotypes conrming the compositional variability of the
essential oils of this species in relation to its geographical origin
and vegetation cycle (Charles and Simon, 1991; Lawrence, 1998;
Bianchini et al., 2001; Angioni et al., 2003; Paolini et al., 2006). At
least three chemical compositional proles have been evidenced
from this study: (a) genotypes rich in nerol and its esters (I); (b)
genotypes with a dominance of - and -selinene (II); (c) geno-
types with high amounts of -curcumene (III). Chemical prole I of
H. italicum ssp. italicum plants resembles that previously reported
for the essential oils from the aerial parts of plants collected in
other locations in Italy or in Corsica, with the difference that, in
those studies, nerol and its esters were found to be often asso-
ciated with good quantities of -curcumene (Charles and Simon,

Fig. 5. In vitro establishment of H. italicum ssp. italicum after 30 days from transplant in the greenhouse.
648 I. Morone-Fortunato et al. / Industrial Crops and Products 32 (2010) 639649
1991; Lawrence, 1998; Bianchini et al., 2001; Angioni et al., 2003;
Paolini et al., 2006).
Nerol and its esters have also been found as characteristic com-
ponents of the essential oil of owers from some genotypes of H.
italicum ssp. microphyllum (Satta et al., 1999; Perrini et al., 2009b)
averaging 4050% of the total oil components. Based on the avail-
able chemical data in the literature (Bianchini et al., 2001; Satta et
al., 1999; Paolini et al., 2006; Perrini et al., 2009), identication and
quantication of the botanical origin of the essential oils or their
derived products from one or the other subspecies based only on
the chemistry might not be appropriate in the case of Helichrysum
populations sharing chemotype I.
Chemotypes II and III appear new for H. italicum ssp. italicum. The
two sesquiterpenes - and -selinene (chemotype II) have not been
identied previously as typical dominant constituents of the essen-
tial oils from this subspecies. -Curcumene, identies genotypes of
chemotype III, and is associated with high amounts of -selinene
(Table 4). In contrast to previous reports (Charles and Simon, 1991;
Lawrence, 1998; Bianchini et al., 2001; Angioni et al., 2003; Paolini
et al., 2006), the content of nerol and its esters in genotypes of
both chemotypes II and III, compared to genotypes belonging to
chemotype I, appears instead greatly reduced (Table 4).
Within the subspecies of H. italicum, -diketones are consid-
ered especially characteristic of H. italicum ssp. italicum (Tira and Di
Modica, 1967; Manitto et al., 1972). This is also conrmed by results
of the current study (Table 4) and previous ndings on H. italicum
ssp. microphyllum (Perrini et al., 2009b). Interestingly, genotypes
with the highest content and the larger number of -diketones
are those belonging to chemotype I. In contrast, -diketones are
present in very low amounts in those genotypes accumulating
sesquiterpenes in their oils (Table 4).
Presence of the epi-eudesmol rosifoliol is reported as variable
in H. italicum ssp. michrophyllum (Satta et al., 1999; Bianchini et
al., 2001; Perrini et al., 2009b), but has never been detected in H.
italicum ssp. italicum, probably due to a misidentication with other
eudesmol compounds. Discrete amounts of rosifoliol were found in
some of the genotypes (5, 8, 11, 15, 19, 23 and 27), always associated
with other eudesmol derivatives (Table 4).
The nding of different chemotypes of H. italicum ssp. italicum
as described here should also be regarded as interesting for a
diversied industrial utilization of the plant, beside its applica-
tion in phytotherapy. Nerol and its derivatives, for example, are
largely employed as cosmetic ingredient for their sweet rose fra-
grance, while curcumene derivatives or drugs containing them are
mainly used in the food industry (Ansari and Curtis, 1974). Alfa-
and -selinene have, respectively, a sweet woody and herbaceous
fragrance and they have acquired importance in chemical ecology
as pheromones (Harborne, 1993; Breitmaier, 2006).
All 20 genotypes subjected to genetic characterization with
110 AFLP polymorphic bands disclosed a clear genetic distinc-
tion, besides their chemical diversity. The phenetic dendrogram
(Fig. 4) showed a clustering among the 20 genotypes that reects
the chemical characterization suggesting that the analysis using
AFLP markers might provide the evidence for some genomic regions
linked to loci involved in the expression of genes coding for chem-
ical compounds that were identied in the investigated essential
oils. In the literature are present several works regarding the chem-
ical analysis and antimicrobial activity of Helichrysum (Satta et al.,
1999; Barbern et al., 1990; Szgeca et al., 2005; Perrini et al.,
2009b), but very few that combine the phenotype and genotype
analysis (Scialabba et al., 2008; Perrini et al., 2009a).
The dendrogram includes three main clades, two of them are
linked at a genetic distance of 0.50 in one main branch (cluster 1
shared in a and b), while the other one appears clearly separated
in the phenetic tree at the same distance (cluster 2). Furthermore,
the main branch, indicated with the number 1 is divided up into
two minor clades: one including genotypes 2, 5, 6, 3, 7, and 8; the
other including genotypes 9, 10, 13, 16, 19, 20, 11, 18, and 27. The
second cluster labelled as 2 includes the remaining genotypes
14-15-23-17-26. The mean genetic similarity among the genotypes
was 0.75. No ambiguous cases of homonymy were found.
As described above, the chemical analysis has differentiated the
essential oils from the 20 genotypes of H. italicun ssp. italicum into
three principal chemotypes, that are also reected in the genetic
study. Among genotypes classied as extremely rich in nerol and
its esters (Table 4), the genotypes 3, 5 and 7 are grouped together
in the rst subgroup of cluster 1, while genotype 23 is separated
at a distance of 0.58 and located in cluster 2. In agreement with
the chemical analysis, genotypes characterized by a prevalence of
both - and - selinene are all grouped in the cluster number 1.
Very interestingly, genotypes distinguished by a high amount of
either -selinene or -selinene appear separated in the cluster,
the rst group of genotypes (10, 11, 19) included in one of the
branches inside clade 1, the others (14, 15) in clade 2. More-
over, the unique genotype 17, with a high amount of -bisabolene
together with -curcumene, forms an independent branch inside
group 2. Similarly, genotype 26 characterized by a chemical pro-
le of the essential oils, difcult to include in the above dened
chemotypes, forms another independent branch inside clade 2,
close to genotype 17 at a distance of 0.58.
Finally, data described in this paper show that the micro-
propagation protocol previously established for H. italicum ssp.
microphyllum (Perrini et al., 2009b) is also suitable for the micro-
propagation of H. italicum ssp. italicum. The growth conditions
adopted in this study allowed us to obtain in vitro clones of H.
italicum ssp. italicum corresponding to the mother plant in terms
of histological, caryological and morphological characteristics.
Adaptability of the 20 genotypes, although always positive, pro-
duced some variable responses in the in vitro growing conditions
(Tables 6 and 7), likely determined by genetic differences between
the genotypes (Hartmann et al., 2002; Pecaut and Martin, 1992).
5. Conclusion
Our observations demonstrate that even if the genetic char-
acters inuence the essential oil composition, they are not a
determinant in changing the phenotypic traits. In this work, an ef-
cient micropropagation protocol was established for H. italicum ssp.
italicum. The protocol assures that the genetic clones are identical
to the mother plant (a selected genotype).
The constitution of micropropagated clones ensures uniformity
of the regenerated material and the essential oils yield and quality
(Noguiera and Romano, 2002; Kalemba and Thiem, 2002; Bertoli et
al., 2004; Perrini et al., 2009b; Avato et al., 2005; Morone-Fortunato
and Avato, 2008) and allows the possibility to use H. italicum ssp.
italicum in the industrial sector.
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