Raluca_163-72_2015

63
Propagation of Ornamental Plants
Vol. 15, № 2, 2015: 63-72
Received: April 2, 2015 Accepted: May 25, 2015
Introduction
Drought and salinity are the abiotic factors that have
the most devastating effects on horticulture. Insufficient
rainfall induces a progressive reduction of the amount
of water available for plants in the soil, which further
affects their growth and development, and the produc-
tivity of crops (Jabeen and Ahmad 2011). Irrigation
is required to mitigate drought stress, but prolonged
irrigation causes secondary soil salinisation, which is
responsible for the yearly loss of more than 10 mil-
lion ha of arable land (Owens 2001). These losses are
expected to increase in the years ahead because of the
foreseeable effects of climate change, which could re-
duce agricultural yields by more than 50% in the next
two decades (Lobell et al. 2008). For this reason, there
is an urgent need to use new strategies for cultivation of
plants, including ornamental species. Besides applying
water-saving techniques, the use of alternative irrigation
water sources should be considered for certain species
(Valdez-Aguilar et al. 2009). As this will generally
imply the use of water with a higher salt content such
as sewage, industrial and agricultural waste water, or
brackish water from saline aquifers the selection of
more stress tolerant cultivars is getting considerable
attention.
Simulation of drought or salinity in open-air field
conditions may be difficult, whereas in vitro experi-
ments avoid technical difficulties and allow the screen-
ing of a larger number of varieties. One of the most
easily achieved and reliable testing for plant stress
tolerance can be carried out at the seed germination
Comparative analysis of osmotic and ionic stress effects on seed
germination in Tagetes (Asteraceae) cultivars
Raluca Cicevan1
, Mohamad Al Hassan2
, Adriana Sestras1
, Monica Boscaiu3
*, Adrian Zaharia1
,
Oscar Vicente2
, and Radu Sestras1
1
University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca,
3-5 Manastur str., 400372 Cluj-Napoca, Romania
2
Institute of Plant Molecular and Cellular Biology (IBMCP, UPV-CSIC), Polytechnic University of Valencia,
CPI, Building 8E, Camino de Vera s/n, 46022 Valencia, Spain
3
Mediterranean Agroforestry Institute (IAM, UPV), Polytechnic University of Valencia, CPI, Building 8E,
Camino de Vera s/n, 46022 Valencia, Spain,
*Fax: + 34-96-387 92 69, *E-mail: mobosnea@eaf.upv.es
Abstract
Drought and salinity are the most adverse environmental factors affecting crop productivity worldwide. The
increasing scarcity of fresh, high-quality irrigation water is forcing the use of alternative water sources-such
as sewage, waste water or brackish water in agriculture, in general, and also in floriculture. Seed germination
is the first and the most important stage in a plant’s life cycle, but it is also the most sensitive to environmental
conditions, representing a bottleneck in plant development. Checking for stress tolerance at the germination
stage is a reliable approach for the rapid screening of a large number of plant cultivars. The aim of the present
study was to analyze the responses to osmotic and ionic stress, induced by isotonic solutions of polyethylene
glycol (PEG) and NaCl, in 13 cultivars of Tagetes patula, T. tenuifolia, and T. erecta at the germination stage.
Germination percentages and rates, and radicle, hypocotyls, and cotyledon lengths were determined after
seven days of treatment. Responses to osmotic stress induced by PEG were similar in all selected cultivars,
but significant differences were detected upon the salt treatments. In general, T. erecta responded better to salt
stress than the other two species, but a large variability among cultivars within each species was observed. The
most stress-tolerant cultivars of T. patula were ‘Orion’and ‘Robuszta’, and those of T. tenuifolia were ‘Luna
Gold’, ‘Luna Rot’ and ‘Luna Orange’.
Key words: genotypes, osmotic stress, salt stress, Tagetes erecta, T. patula, T. tenuifolia

64
stage (Richards 1978). Germination is the first and
most important phase in the plant life cycle, which is
decisive for survival, but it is also the most sensitive
to environmental stressful conditions. Germination in-
volves a complex series of physiological events, which
starts with the uptake of water by seeds and terminates
with the elongation of the embryonic axis (Bewley
1997). Water availability plays a major role in germi-
nation, as water is necessary for enzymatic reactions,
solubilisation and transport of metabolites, and also
as a reagent in the hydrolytic biochemical processes
that occur during germination (Muscolo et al. 2014).
Inadequate levels of soil moisture at sowing may not
only completely prevent seed germination, but can also
induce an irregular seedling emergence with negative
effects on further plant development (Passioura 2002).
Among the many environmental factors that in-
fluence seed germination, this process is extremely
sensitive to soil salinity. Even for most halophyte spe-
cies that naturally grow in highly saline habitats seed
germination is optimal under non-saline conditions,
and it is strongly affected even by salt concentrations
much lower than those tolerated by adult plants (Ungar
1995). Seed of halophytes usually develop the abil-
ity of dormancy during the periods of stress and are
ready to germinate when salinity is alleviated and the
environmental conditions are appropriate for seedling
growth (Gul et al. 2013). In glycophyte salt-sensitive
plants germination is often inhibited by very low salt
concentrations. Salinity has two major effects of plants:
osmotic stress and ion toxicity. The osmotic component
results from dehydration and loss of turgor induced by
external solutes. This effect is not specific for high soil
salinity, but it is also induced by other abiotic stresses
such as drought, cold or high temperatures (Greenway
and Munns 1980). The second component of salt stress,
ion toxicity, occurs at high intracellular concentrations
of Na+
and Cl-
, which inhibit the activity of many en-
zymatic systems and some fundamental cellular proc-
esses, such as protein synthesis or mRNA processing
(Zhu 2001, Forment et al. 2002). Salinity also affects
plant nutrition by competition of Na+
with the uptake
of essential cations, especially K+
and Ca2+
(Zhu 2001).
Among additional deleterious effects, NaCl also modi-
fies the contents of plant growth regulators, including
an increase in abscisic acid (ABA). This could partially
explain the high sensitivity of seed germination to salt
stress, as ABA inhibits germination (Zhu 2001).
The aims of the present study were: (a) to determine
the germination response of 13 marigold cultivars to
stress in the presence of NaCl and PEG, differentiat-
ing the osmotic effect from ion toxicity, (b) to evaluate
the effects of NaCl and PEG on the lengths of radicle,
hypocotyl, and cotyledons at early seedling stages, and
(c) to select the most tolerant cultivars, which will prob-
ably grow better under water and salt stress conditions.
Materials and Methods
Plant material
Seeds of 13 cultivars belonging to three different
Tagetes species were used in this study: French mari-
gold, Tagetes patula (‘Orion’, ‘Robuszta’, ‘Bolero’,
‘Orange Flame’, and ‘Szinkeverek’); golden or signet
marigold, Tagetes tenuifolia (‘Sarga’, ‘Luna Gold’,
‘Luna Lemon’, ‘Luna Rot’, and ‘Luna Orange’); and
African marigold, Tagetes erecta (‘Magas Citromsarga’,
‘Cupid Golden Yellow’, and ‘Alacsony Citromsarga’).
Seed germination
Seed germination was conducted in Petri dishes
(diameter 9 cm) on double filter paper discs and cotton
moistened with 15 ml of distilled water (for the con-
trols) or the PEG and NaCl solutions, and sealed with
parafilm to prevent evaporation. Seeds were germinated
under different osmotic potentials, –0.25 MPa, –0.5
MPa, and –1 MPa, induced either by PEG (6000) or
by NaCl. Distilled water was used as control (0 MPa).
PEG concentrations were calculated according to the
equation of Michel and Kaufmann (1973):
Ψh = – (1.18 × 10-2
) × C – (1.18 × 10-4
) × C2
+ (2.67 ×
10-4
) × CT + (8.39 × l0- 7
) × C2
T,
where Ψh: water potential (MPa); T: temperature in
°C; and C: PEG-6000 concentration in the solution (g l-1
).
For the salt-stress treatments, NaCl concentrations
of equivalent osmotic pressure to those with PEG were
calculated based on the Van’t Hoff’s equation (Ben-Gal
et al. 2009), which resulted in the following salt con-
centration: 57.2 mM NaCl (equivalent to –0.25 MPa),
114.6 mMNaCl (equivalent to –0.5 MPa), and 227.8
mM NaCl (equivalent to –1 MPa).
Four replicates of 25 seeds were used for each treat-
ment and each cultivar. Germination was performed in
a germination chamber at 23°C, with 16 hours photope-
riod. The number of germinated seeds was recorded
daily, and seeds were considered as germinated if the
radicle protruded at last 2 mm. Germination capacity
(%) was determined after seven days and the germina-
tion rate was calculated as MGT (mean germination
time) according to the formula provided below (Ellis
and Roberts 1981):
MGT= Σ D n/Σn,
Where "D" is the days from the beginning of the
germination test, and "n" is the number of seeds newly
germinated on day "D".
Analysis of seedling growth
On the seventh day from the beginning of ex-
periment, radicle, hypocotyl, and cotyledonary leaves
lengths were measured after scanning the seedlings,
using ‘imageJ’software (Rasband 1997-2012). The re-
duction of seedling length was calculated as percentages
Raluca Cicevan et al. Effect of PEG and NaCl on seed germination in Tagetes

65
with respect to the values registered in the correspond-
ing control, for each cultivar (Table 1). Seedling vigour
index (SVI) was calculated by the following formula
(Abdul-Baki and Anderson 1973):
SVI = Germination (%) × [Mean root length (mm)
+ Mean hypocotyl length (mm)].
Statistical analysis
The statistical analysis was performed using the
SPSS 16 statistical programme. Prior to analysis of vari-
ance, germination percentages were arcsine transformed
and reduction of radicle, hypocotyl, and cotyledon was
calculated in percentages, considering as 100% their
length in control treatments. The post hoc Duncan test
was applied to identify the homogenous groups when
the ANOVA null hypothesis was rejected (p < 0.05).
Results
The germination was higher than 80% (Figs. 1 and
2) and was completed within seven days (Figs. 3 and 4)
Fig. 1. Seed germination (%) of different cultivars of three Tagetes species in the presence of PEG (polyethylene glycol) at
the indicated osmotic potentials.
Means ± standard deviation (SD) followed by the same lowercase letter were not significantly different among treatments, and
means followed by the same capital letter were not significantly different among cultivars, according to the Duncan’s test (p < 0.05).
Cultivar abbreviations: Or - ‘Orion’, Ro - ‘Robuszta’, Bo - ‘Bolero’, Of - ‘Orange Flame’, and Sz - ‘Szinkeverek’ (Tagetes
patula); Sa - ‘Sarga’, Lg - ‘Luna Gold’, Ll - ‘Luna Lemon’, Lr - ‘Luna Rot’, and Lo - ‘Luna Orange’ (T. tenuifolia); Mc -
‘Magas Citromsarga’, Cg - ‘Cupid Golden Yellow’, and Ac - ‘Alacsony Citromsarga’ (T. erecta).
T. patula
aAB aAB
bB
aB
aA
aAB aB aB
aA
aB aA cA
aAaBC
aA
bC
aABC
aAB
aAB
aAB
aB
aA
aB aB
bcA
aB
aAB
aA
aAB
aAB aB
aA
aA
aA
aA
aA
aB
bA
aB
aABC
aA
bC
aBC
aAB
aA
aA aA
aA aA
aB
aA
aB
0
20
40
60
80
100
120
Or Ro Bo Of Sz Sa Lg Ll Lr Lo Cg Al Mc
Control -0.25 Mpa -0.5 Mpa -1Mpa
T. patula T. tenuifolia T. erectaT. tenuifolia T. erecta
Germination(%)
Fig. 2. Seed germination (%) of different cultivars of three Tagetes species in the presence of the indicated concentrations on NaCl.
cAB cAB
cB
cB
bA
cAB cB cB
cA
cB
bA
cA
cA
cAB
cAB
cC
bcBC
bA
bA
cBC cC
bcAB
bcBC
bA
bcA cA
bB
bA
bB
bB
aA
abA
bA
bA
bA
bA
bB
bA
bA
aAB
aA
aB
aAB
aB
aAB
aAB
aA
aAB
aB
aA
aA
0
20
40
60
80
100
120
Control 57.2 mM NaCl 114.4 mM NaCl 228.8 mM NaCl
T. patula T. tenuifolia T. erectaT. patula T. tenuifolia T. erecta
Germination(%)
Vol. 15, № 2, 2015: 63-72

66
in all cultivars in the control treatment. The highest ger-
mination percentage (100%) was recordered in T. erecta
‘Cupid Golden Yellow’ and ‘Alacsony Citromsarga’,
and the lowest (72.5%) in T. patula ‘Szinkeverek’. Op-
timal germination in the T. patula and T. erecta cultivars
was recorded in control treatments in the absence of
stress, and the presence of PEG resulted in lower mean
germination percentages. The situation was somewhat
different for the T. tenuifolia cultivars (except ‘Luna
Orange’), in which the highest mean germination per-
centages were observed in seeds exposed to the highest
PEG concentration tested (Fig. 1). The effect of NaCl
on seed germination (Fig. 2) was clearly stronger than
that of PEG. Under the highest concentration of NaCl
tested, less than half of the seeds germinated, with the
exception of ‘Cupid Golden Yellow’ of T. erecta (52%
germination). Even at lower concentrations, NaCl
had a clearly inhibitory effect on germination of most
cultivars, although T. erecta varieties appeared to be
relatively more resistant to salt stress.
MGT increased when seeds were treated with PEG
or with NaCl, in a concentration-dependent manner and
for all tested cultivars; that is, the stress treatments led
to slower germination. This retardation of germination
was relatively stronger in the presence of NaCl than in
the presence of PEG (Figs. 3 and 4).
The effect of the stress treatments on seedling de-
velopment was more evident than on seed germination.
PEG treatments induced a concentration-dependent
reduction on radicle and hypocotyl lengths, in all cul-
tivars under study (Tables 2 to 4), although this effect
was relatively weaker in the case of T. erecta cultivars
Fig. 3. Seed mean germination time (MGT) of different cultivars of three Tagetes species in the presence of PEG at the indi-
cated osmotic potentials
Fig. 4. Seed mean germination time (MGT) of different cultivars of three Tagetes species in the presence in the presence of
the indicated concentrations on NaCl
aB
aAB
aA
aB
aC
bAB
abAB
aA
aB
aA
aA
aA
aB
bB
bBC
abA
bB
aC
abBC
aA
abB
abC
abA
bA
bAB abB
bB abA
abcA
bB
aC
abB
abA abA
bB
abcA
bcA
bcAB
bB
bAB
bB
cA
bAB
aB bB
bA
bA
abAB
cB
cA
cAB
bB
0
1
2
3
4
5
Control -0.25 Mpa -0.5Mpa -1Mpa
Meangerminationtime(days)
aB
aAB
aA
aB
bC
aB
aB
aA
aB
aA
aA
aA
aB
abC
bC
abA
abB
aB
bC
abAB
bA
bB
bA
bA
bA
bB
bC
cD
bA
cB
bB
cC
abcAB
bcAB
cB
bA
bcA cA
bB
bC
bC
cB
bA
bAB
cAB
cB
cAB
cA
cA
dB
cC
0
1
2
3
4
5
Meangerminationtime(days)

67
(Table 4), as compared to the other two species. Re-
garding cotyledons lengths, the decreasing trend in
the presence of PEG was generally maintained, but
the relative reduction was less marked than in the case
of radicle or hypocotyl length. In fact, the differences
between treatments were not statistically significant in
T. erecta cultivars (Tables 2 to 4).
NaCl treatments also induced a reduction of the
seedlings’ radicle and hypocotyls lengths in all tested
cultivars; as in the case of PEG treatments, the cotyle-
don length was not so much affected by salt, at least at
the lowest NaCl concentration. Moreover, here again
a relatively higher resistance was observed in the T.
erecta cultivars than in the other two Tagetes species
Table 1. Mean radicle, hypocotyls, and cotyledon length (mm) in control treatments in the absence of stress.
Species Cultivar Radicle Hypocotyl Cotyledon
T. patula
Orion 4.5 ± 0.3 c 1.4 ± 0.6 b 0.5 ± 0.0 bc
Robuszta 4.0 ± 0.2 bc 1.4 ± 0.1 b 0.5 ± 0.0 c
Bolero 3.9 ± 0.9 bc 1.1 ± 0.1 a 0.4 ± 0.0 a
Orange Flame 4.0 ± 0.5 c 1.1 ± 0.1 a 0.5 ± 0.0 bc
Szinkeverek 3.3 ± 0.3 a 1.3 ± 0.7 b 0.5 ± 0.0 b
T. tenuifolia
Sarga 3.1 ± 0.3 a 0.9 ± 0.0 c 0.3 ± 0.0 b
Luna Gold 2.9 ± 0.2 a 1.1 ± 0.1 c 0.3 ± 0.0 a
Luna Lemon 3.2 ± 0.2 a 0.8 ± 0.1 b 0.3 ± 0.0 a
Luna Rot 3.6 ± 0.3 a 0.6 ± 0.1 a 0.3 ± 0.1 a
Luna Orange 3.5 ± 0.2 a 0.8 ± 0.0 b 0.3 ± 0.0 a
T. erecta
Cupid Golden Yellow 2.6 ± 0.2 a 0.6 ± 0.0 a 0.5 ± 0.0 a
Alacsony Citromsarga 3.0 ± 0.2 b 0.7 ± 0.0 a 0.4 ± 0.0 a
Magas Citromsarga 3.6 ± 0.2 c 1.0 ± 0.1 b 0.5 ± 0.0 b
Means ± standard deviation (SD) followed by the same letter in each column did not differ significantly, according to the Duncan’s
test (p < 0.05).
Table 2. Effects of isotonic PEG and NaCl solutions on young seedlings of Tagetes patula.
Radicle length (% of control)
Cultivar
–0.25 MPa –0.5 MPa –1 MPa·
PEG NaCl PEG NaCl PEG NaCl
Orion 70.5 ± 3.2 e AB 25.5 ± 2.4 c B 66.6 ± 3.2 e B 8.3 ± 0.3 b B 52.3 ± 4.2 d C 0 a A
Robuszta 57.0 ± 5.3 e A 24.1 ± 1.1 c B 52.3 ± 4.6 e A 13.1 ± 0.5 b C 39.2 ± 3.6 d AB 0 a A
Bolero 76.6 ± 3.3 c B 14.7 ± 0.1 a A 72.9 ± 4.9 c B 0.0 ± 0.0 a A 31.3 ± 3.8 b A 0 a A
Orange Flame 63.8 ± 3.2 e AB 14.4 ± 1.0 b A 54.2 ± 3.5 d A 0.0 ± 0.0 a A 47.4 ± 2.0 c BC 0 a A
Szinkeverek 77.8 ± 7.3 e B 22.8 ± 1.9 b B 62.3 ± 3.4 d AB 0.0 ± 0.0 a A 46.4 ± 3.2 c BC 0 a A
Hypocotyl length (% of control)
Orion 92.5 ± 2.7 e B 76.0 ± 5.0 c B 87.1 ± 3.6 de B 31.0 ± 1.7 b B 82.5 ± 3.7 ed B 0 a A
Robuszta 78.7 ± 4.0 d A 60.6 ± 4.8 c A 72.7 ± 3.4 d A 32.2 ± 3.0 b B 60.4 ± 3.1 c A 0 a A
Bolero 94.8 ± 4.8 c B 80.1 ± 4.2 b B 80.7 ± 5.0 b AB 0.0 ± 0.0 a A 68.9 ± 6.3 b B 0 a A
Orange Flame 94.9 ± 5.8 c B 78.8 ± 5.2 b B 89.3 ± 5.5 bc B 0.0 ± 0.0 a A 85.1 ± 4.0 bc A 0 a A
Szinkeverek 86.3 ± 3.0 c A 77.5 ± 3.7 c B 85.3 ± 3.8 c A 0.0 ± 0.0 a A 64.4 ± 5.2 b A 0 a A
Cotyledon length (% of control)
Orion 96.2 ± 3.0 cd B 113.2 ± 4.8 e A 92.5 ± 2.1 c B 100.9 ± 2.4 d B 82.0 ± 1.4 b B 0 a A
Robuszta 94.0 ± 3.5 c B 103.4 ± 2.6 d A 79.7 ± 2.1 b A 99.9 ± 8.7 d B 79.0 ± 2.7 b AB 0 a A
Bolero 95.4 ± 4.3 c B 135.2 ± 4.8 d B 79.3 ± 2.4 b A 0.0 ± 0.0 a A 73.5 ± 3.3 b A 0 a A
Orange Flame 81.6 ± 2.3 c A 111.8 ± 4.1 c A 75.6 ± 2.7 c A 0.0 ± 0.0 a A 76.3 ± 2.1 c AB 0 a A
Szinkeverek 95.0 ± 3.1 c B 127.3 ± 4.0 d B 95.4 ± 2.7 c B 0.0 ± 0.0 a A 79.7 ± 1.7 b AB 0 a A
Means ± standard deviation (SD) followed by the same lowercase letter in each row were not significantly different among treat-
ments, and means followed by the same capital letter in each column were not significantly different among cultivars, according
to the Duncan’s test (p < 0.05).
Vol. 15, № 2, 2015: 63-72

68
(Tables 2 to 4). The inhibition of seedling growth
by NaCl was generally stronger than that observed
in the presence of isoosmotic PEG concentrations.
In fact, under the lowest osmotic potential (-1MPa;
227.8 mM NaCl) and, for several cultivars, also at
the intermediate salt concentration tested (-0.5 MPa,
114.6 mM NaCl), seedlings measurements were not
possible (‘0’ values in Tables 2 to 4) since, although
the radicle emerged in some seeds, seedlings did not
develop further.
The most illustrative deleterious effect of osmotic
and ionic stress was shown by the reduction of the
seedling vigor index (SVI, Figs. 5 and 6). PEG treat-
ments caused a significant, concentration-dependent
reduction of SVI (Fig. 5), and the effect of NaCl, at
the same isoosmotic concentrations, was by far more
Table 3. Effects of isotonic PEG and NaCl solutions on young seedlings of Tagetes tenuifolia.
Cultivar
–0.25MPa –0.5MPa –1MPa·
Sarga 70.7 ± 5.6 c AB 12.8 ± 0.9 a A 67.0 ± 5.5 c A 0.0 ± 0.0 a A 35.7 ± 4.4 b B 0 a A
Luna Gold 88.4 ± 6.8 d C 19.5 ± 1.2 b BC 63.3 ± 6.5 c A 13.4 ± 0.9 b C 58.5 ± 4.9 c C 0 a A
Luna Lemon 66.6 ± 6.0 e AB 15.5 ± 1.3 b AB 51.6 ± 3.8 d A 0.0 ± 0.0 a A 31.7 ± 3.3 c B 0 a A
Luna Rot 52.2 ± 5.2 d A 20.4 ± 1.3 c BC 52.7 ± 5.6 d A 8.6 ± 0.7 b B 18.1 ± 1.2 c A 0 a A
Luna Orange 78.3 ± 5.3 c BC 24.1 ± 2.3 b C 65.7 ± 6.5 d A 7.5 ± 0.5 a B 36.4 ± 2.6 c B 0 a A
Sarga 63.0 ± 5.7 c A 42.0 ± 3.2 b A 60.3 ± 9.2 c A 0.0 ± 0.0 a A 61.4 ± 6.2 c A 0 a A
Luna Gold 84.2 ± 6.4 d B 65.0 ± 3.7 c B 81.3 ± 6.0 d B 41.2 ± 2.3 b B 54.4 ± 3.4 c A 0 a A
Luna Lemon 79.0 ± 4.8 b AB 74.3 ± 3.4 b B 73.6 ± 5.8 b AB 0.0 ± 0.0 a A 67.1 ± 5.6 b AB 0 a A
Luna Rot 100.7 ± 7.4 c C 137.7 ± 5.0 d D 103.4 ± 5.3 c C 95.6 ± 7.0 c D 61.5 ± 5.4 b A 0 a A
Luna Orange 84.5 ± 3.5 b B 113.5 ± 8.6 c C 83.1 ± 3.5 b B 73.6 ± 4.5 b C 81.2 ± 4.9 b B 0 a A
Sarga 94.0 ± 3.1 c AB 134.0 ± 5.1 d B 76.8 ± 3.2 b A 0.0 ± 0.0 a A 78.6 ± 4.5 b A 0 a A
Luna Gold 84.4 ± 3.7 b A 113.7 ± 4.0 c A 78.6 ± 4.1 b AB 124.1 ± 0.0 c B 77.1 ± 3.1 b A 0 a A
Luna Llemon 94.8 ± 2.8 c AB 124.1 ± 4.2 d AB 85.3 ± 2.9 c AB 0.0 ± 0.0 a A 68.8 ± 2.4 b A 0 a A
Luna Rot 98.4 ± 4.5 c B 135.4 ± 4.6 e B 87.4 ± 4.5 c B 119.9 ± 8.8 d B 97.7 ± 3.5 c A 0 a A
Luna Orange 99.6 ± 3.9 d B 114.5 ± 4.1 e A 85.3 ± 2.6 c AB 113.9 ± 4.5 e B 73.0 ± 2.9 b A 0 a A
Fig. 5. Seedling vigour index (SVI) of different cultivars of Tagetes patula, T. tenuifolia, and T. erecta, in the presence of PEG
at the indicated osmotic potentials
cB
bAB
cB cB
cA
cB cBC dBC
bA
cC
bB
aA
cCbB
aA
bB
bA bA
bA
bBC
cAB
bA
bC
bB
aA
bB
abC
aA
aBC
abAB bAB
bB
abB
bA
bBC
bC
aB
aA
aB
aD
aA
aCD aBC
aAB
aB
aE
aC
aA
aD
aB
aA
bC
0
1000
2000
3000
4000
5000
6000
Control -0.25MPa -0.5MPa -1MPa
T. patula T. tenuifolia
T.erectaT. patula T. tenuifolia T. erecta
Seedlingvigourindex

69
pronounced (Fig. 6). All cultivars behaved in a simi-
lar manner, qualitatively, except T. erecta ‘Alacsony
Citromsarga’, which showed anomalous reduced SVI
values in the control, non-treated seedlings.
Table 4. Effects of isotonic PEG and NaCl solutions on young seedlings of Tagetes erecta.
Cultivar
–0.25MPa –0.5MPa –1MPa·
Cupid Golden
Yellow
98.9 ± 7.5 e C 27.4 ± 1.6 c B 80.7 ± 5.8 d B 12.3 ± 0.5 b B 75.7 ± 3.6 d B 0 a A
Alacsony
Citromsarga
76.5 ± 6.7 d B 22.4 ± 0.9 b A 62.0 ± 3.5 c A 12.4 ± 0.8 b B 61.0 ± 1.3 c A 0 a A
Magas
Citromsarga
56.1 ± 3.2 d A 19.8 ± 1.3 c A 55.3 ± 4.8 d A 8.4 ± 0.3 b A 64.6 ± 4.4 e A 0 a A
Cupid Golden
Yellow
92.6 ± 5.7 c A 107.9 ± 5.5 d B 84.5 ± 3.9 bc A 73.0 ± 5.8 b B 83.4 ± 3.3 bc AB 0 a A
Alacsony
Citromsarga
104.1 ± 5.2 c A 129.5 ± 4.0 d C 93.2 ± 3.91 c A 66.3 ± 7.3 b B 92.7 ± 3.1 c B 0 a A
Magas
Citromsarga
84.7 ± 6.8 cd A 93.1 ± 4.8 d A 81.0 ± 3.9 cd A 47.9 ± 2.1 b A 73.3 ± 4.4 c A 0 a A
Cupid Golden
Yellow
102.5 ± 3.9 b B 118.5 ± 4.9 c A 94.0 ± 4.1 b A 102.2 ± 4.2 b B 93.1 ± 3.4 b A 0 a A
Alacsony
Citromsarga
101.6 ± 2.3 c B 132.9 ± 5.2 e A 89.8 ± 2.9 b A 117.3 ± 4.7 d C 110.2 ± 3.0 c dB 0 a A
Magas
Citromsarga
90.9 ± 3.5 b A 120.8 ± 3.8 c A 93.4 ± 3.3 b A 88.1 ± 3.3 b A 89.1 ± 2.6 b A 0 a A
Fig. 6. Seedling vigour index (SVI) of different cultivars of Tagetes patula, T. tenuifolia, and T. erecta, in the presence of the
indicated concentration of NaCl
cB
cAB
bB bB
bA
bB cBC bBC
cA
cC
cB
aA
cC
bB
bA aA
aA aA
aA
bB aB bB
bC
bA bAB
bB
aA aA
aA aA
aA aB
aA aA
0
1000
2000
3000
4000
5000
6000
T. patula T. tenuifolia T. erecta
T. patula T. tenuifolia T. erecta
Seedlingvigourindex
Vol. 15, № 2, 2015: 63-72

70
Discussion
Several well-known mechanisms mediate the inhibi-
tory effect of salinity on seed germination and early
seedling development and vigor. As indicated in the
Introduction, among many other deleterious effects, salt
stress includes an osmotic component, altering water
imbibition by seeds, and anion toxicity component,
which inhibits enzymatic activities and basic cellular
processes and directly affects macromolecular struc-
tures (Yupsanis et al. 1994, Dantas et al. 2007).
One of the methods used to mimic drought condi-
tions is the exposure of seeds to PEG 6000, an inert,
non ionic and impermeable compound (Hohl and Peter
1991). Because PEG does not enter the apoplast, water
is withdrawn from the cell and the cell wall. On the other
hand, by using isotonic solutions of PEG and NaCl, the
toxic effect of the salt can be distinguished from its
osmotic component, thus achieving a more complete
analysis of the responses to salt stress.
Most horticultural crops are glycophytes (Greenway
and Munns 1980), plants which grow optimally in the
absence of salt. Even though marigolds are not true
halophytes, they are considered as relatively resistant
to low or moderate salinity. Valdez-Aguilar et al. (2009)
reported that the species T. erecta and T. patula could
withstand soil salinity levels of about EC =3.6 dSm-1
.
Sayyed et al. (2014) showed that T. erecta is moderately
tolerant to salt stress, reporting a significant increase in
plant biomass after two weeks of treatment with 50 or
100 mM NaCl, but a decrease in the presence of higher
(150 mM and 200mM NaCl) salt concentrations. In
gardening, many marigold varieties are also considered
among (relatively) drought tolerant ornamental plants.
Lin et al. (2014) reported that germination rate of spe-
cies Leymus chinensis was significantly affected by pH,
salinity and their interactions. At 200 mM salinity, the
germination percentage was zero.
Results of the in vitro germination assays reported
here support the aforementioned considerations. Seeds
of several of the Tagetes cultivars under study germi-
nated even better than those of the halophyte Plantago
crassifolia under salt stress conditions (Vicente et al.
2004), and better than seeds of two stress-tolerant
Iberian species of the genus Gypsophila (Soriano et
al. 2014) in the presence of both, NaCl and PEG. The
observed inhibitory effects of osmotic and ionic stress
were more pronounced on seedling development than
on the mere fraction of germinated seeds, as reported in
other species (Okçu et al. 2005, Meneses et al. 2011).
Not all seedling structures of the Tagetes cultivars were
affected in the same way by stress, as the inhibitory ef-
fects of PEG and NaCl on early seedling growth were
mostly reflected in a reduction of radicle length. This
has been previously reported in other plant species (Al-
mas et al. 2013) and may be associated to reduced cell
division and elongation during germination (Frazer et
al. 1990).Agreater root lenght confers increased resist-
ance to drought and a better capacity to extract water
from soil. Therefore, the ability to develop extensive
root systems in extremely important and may be used
as a convenient trait to select stress tolerant cultivars
(Muscolo et al. 2014).
Several studies have compared PEG and NaCl treat-
ments of germinating seeds. In Phaseolus mungo (Garg
2010) and Helianthus annuus (Luan et al. 2014) PEG
had a stronger effect than NaCl. In the present experi-
ments, however, NaCl toxicity appeared to inhibit seed
germination and seedling growth to a higher degree
than water stress, mimicked by PEG treatments at the
same isoosmotic concentrations than NaCl. Similar
responses were reported in Agropyron elongatum and
Elymus cinereus (Roundy et al. 1985) and in Atriplex
prostrata and A. patula (Katembe et al. 1998), which
also show higher tolerance to osmotic stress.
Comparing responses to abiotic stresses in a rela-
tively high number of related genotypes represents a
useful strategy in plant breeding. For this reason
selection of plants with improved drought and/or salt
tolerance will be especially important for the future of
agriculture in many areas of the word, considering the
forecasted effects of climate change (Tuberosa and Salvi
2006). The Tagetes cultivars used in this study showed
clear differences in their responses to osmotic and salt
stress during germination, mostly concerning toxicity of
NaCl, as mentioned above. Overall, T. erecta was found
to be the most tolerant of the three selected species, but
a large variability among cultivars within one species
was detected. Thus, ‘Orion’ and ‘Robuszta’ showed
the highest stress tolerance in T. patula, whereas in T.
tenuifolia ‘Luna Gold’, ‘Luna Rot’ and ‘Luna Orange’
were the most tolerant. These results support the useful-
ness of in vitro germination assays and determination
of seedling growth parameters for a rapid selection of
stress tolerant cultivars in floriculture, even though this
tolerance should be confirmed at later developmental
stages. Such cultivars could then be cultivated using
poor-quality, relatively saline water for irrigation, with-
out compromising the economic value of the product,
as already stated for Tagetes (Valdes-Aguilar et al.
2009) and other ornamentals such as Celosia argentea
(Friedman et al. 2007) and Antirrhinum majus (Carter
and Grieve 2008).
Acknowledgements: Raluca Cicevan is PhD
student at the University of Agricultural Sciences and
Veterinary Medicine Cluj-Napoca. This paper was
published according to the PhD study contract no.
14855 from October 1, 2012, and under the frame of
European Social Fund, Human Resources Develop-
ment Operational Programme 2007-2013, project no.
POSDRU/159/1.5/S/132765. MohamadAl Hassan is a
recipient of an Erasmus Mundus pre-doctoral scholar-

71
ship financed by the European Commission (Welcome
Consortium).
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