Effects of GA3Pregerminative Treatment on Gentiana lutea
L. var. aurantiaca Germination and Seedlings Morphology
Óscar González-López1and Pedro A. Casquero2
1Research Group of Engineering and Sustainable Agriculture, Natural Resources Institute, University of Le´
24071 Le ´
2Research Group of Engineering and Sustainable Agriculture, Department of Agrarian Engineering and Sciences, University of Le´
Ave n ue Po r tu g al 41 , 24 07 1 Le ´
Correspondence should be addressed to Pedro A. Casquero; firstname.lastname@example.org
Received April ; Accepted June ; Published June
Academic Editor: Antonio M. De Ron
Copyright © ´
opez and P. A. Casquero. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
Gentiana lutea L. is widely used in bitter beverages and in medicine; Gentianae Radix is the pharmaceutical name of the root of
G. lutea. ese uses have generated a high demand. e wild populations of Gentiana lutea var. aurantiaca (M. La´
ınz) M. La´
have been decimated; it is necessary to establish guidelines for its cultivation. Gentian as most alpine species has dormant seeds.
Dormancy can be removed by cold and by means of a gibberellic acid (GA3) treatment. However, cold treatments produce low
germination percentages and GA3treatments may produce o-type seedlings. So, the objective was to determine, for the rst time,
the presowing treatments that allow high germination rate and good seedling morphology. e best pregerminative doses of GA3
to break seed dormancy were , , and ppm, while the best doses to optimize the seedling habit were and ppm.
is study provides, for the rst time, a ppm GA3dose that led to a high germination rate and good seedling morphology, as
the starting point for gentian regular cultivation.
Gentiana lutea L. (Gentianaceae) is a herbaceous perennial
plant native to the mountains of central and southern Europe
food products, and also in traditional medicine to stimulate
theappetiteandimprovedigestion[,]. ese uses have
generated a great demand, so that more than tons of
gentian root is produced from tons of wild stocks every
year around the world . Due to the slow growth of this
species,thewildpopulationsofG. lutea have been decimated
and are now close to their disappearance and/or are dicult
to revive. e increasing demand has provoked alarm about
the extinction of the species, and for this reason gentian is
being protected throughout Europe by law.
In the Northwest part of Iberian Pen´
Cantabric Mountains), gentian root has been used as a tonic
for the stomach and to restore appetite . In this area
Gentiana lutea L. owers show a colour ranging from orange
to almost red when compared to yellow owers of G. lutea
L. subsp. lutea. ese populations have been classied as
Gentiana lutea L. var. aurantiaca . In the Le´
some populations of aurantiaca variety populations show
which indicates reduced reproductive tness and elevated
extinction risks .
e main economic sources in European mountain areas
include mining (a decreasing activity), ranching (in crisis),
and tourism. Collection of gentian roots has traditionally
been a supplement to the family income. Due to the current
economic crisis, people have started to collect gentian again
in a furtive way for an extra income. Considering the harsh
climate conditions of the mountain regions, where both
horticulture and fruit growths are dicult to carry out, the
cultivation of gentian is an alternative economic activity for
large unused lands with agro climatic characteristics suitable
for its growth.
Hindawi Publishing Corporation
e Scientiﬁc World Journal
Volume 2014, Article ID 751279, 6 pages
e Scientic World Journal
G. lutea, as most alpine species, has dormant seeds,
exhibiting mainly physiological dormancy [,]. is
dormancy can be removed by cold treatment [,], by
cold stratication , and by GA3treatment. GA3,alone
or together with other chemical or physical treatments, is
widely used to remove seed dormancy ortoimprovethe
development of the seedlings, although diverse results have
been obtained [–]. Related to this, a study with G. lutea
seeds was carried out on germination rates in Petri dishes
using GA3and a stratication treatment . However, some
of these treatments, such as gibberellic acid, may produce
weak and o-type seedlings in G. lutea .
erefore, this study aims to establish which GA3dose,
applied as pregerminative treatment, improves the germina-
tion rate without aecting the development of seedlings, as
the rst step for gentian cultivation.
2. Materials and Methods
2.1. General Seed Trials. Seeds of Gentiana lutea L. var.
aurantiaca from population located in the western part of
the Cantabrian mountains (Spain) were collected (Figure ).
Seeds from mature capsules containing well-developed ripe
seeds were taken from randomly selected plants. Aer this,
the seeds were manually cleaned, removing any damaged,
empty, or visually malformed seeds, kept in paper bags, and
then stored in a fridge at ∘Cinsideadarkglassbottlewith
Stratication Treatment. Seeds were stratied for months
over a silica sand base (Ø .–. mm). Both tray and silica
sand were sterilized by autoclaving at . kg cm−2 and ∘C
for h, on each of three consecutive days. Aer placing the
seeds well distributed over the tray surface, another layer
( cm in height) of silica sand was added covering the seeds
(Figure ). Finally, the sand was moistened with distilled
water, avoiding saturation and placing the tray inside the
refrigerator at ∘C±∘C. e tray was checked every week
during the next days in order to control the sand humidity.
Stratied seeds were cleaned using a metallic sieve with Ø
GA3Tre atm ent. Seeds were soaked at ∘Cforhours
in GA3(Sigma-Aldrich) water solution (, , , and
ppm) and were cleaned with distilled water to remove
GA3residues before sowing.
2.2. Petri Dishes Germination Assay. Tw o t reat ment s were
applied to the gentian seeds: cold treatment (stratied or not)
of seeds in each treatment were tested for germination
on top of a lter paper sheet with ml distilled water in cm
plastic Petri dishes (Figure (b)). Filter papers were rewetted
with distilled water as required. Dishes were checked twice
a week during a -day test period and germinated seeds
were counted and removed. It was established that seeds
were germinated when the length of the radicle exceeded
the seed coat with mm (Figure (c)). e conditions during
60 km Le ́
F : Map showing the location of sampled population of
Gentiana lutea L. var. aurantiaca.
F : Addition of silica sand over gentian seeds for stratication
this time were in a growth chamber (Figure (a)) with a
constant temperature of ∘Candahlightphotoperiod
(provided by cool white uorescent tubes with an irradiance
of 𝜇mol⋅m−2⋅s−1). At the end of the incubation period
( days), the nal germination percentage and the mean
germination time (MGT) were calculated according to Ellis
and Roberts .
2.3. Forest Trays Assay. Five replicates of seeds each were
tested with ve GA3dose (, , , and ppm). All
seeds were stratied as specied in Section ..eforestry
seed trays had a volume of cm3and a height of cm. A
commercial substrate, n∘ from Pindstrup Mosebrug Sae,
black peat (pH .–), with similar conditions to those of the
mountain soils. Pesticide seed treatment (Tiram % WP)
was applied according to Valenciano et al. tocontrolthe
development of phytopathogenic fungi . Every lot of
seeds was sowed in a unique tray and every cell was covered
with cm3of sterilized silica sand, which is equivalent to
days aer sowing, the measurement of the plants
was carried out. Once the development of the plants was
observed, two dierent characteristics were measured: stem
length, that is, the length of the stem (mm) from the substrate
level to the apex of the primary stem, and leaf length, the
length (mm) of the most developed leaf in the third pair
e Scientic World Journal
F : Petri dishes germination assay of G. lutea L. var. aurantiaca: (a) growth chamber; (b) Petri dishes distribution; (c) germinated seeds.
of leaves starting from the base. Both measurements were
performed in random seedlings per replication.
2.4. Statistical Analysis. e values of nal germination
percentages were arcsine transformed (untransformed data
appears in the gures). In each experiment, the data was ana-
lyzed using the general linear model (GLM procedure) and
whenever the analyses showed signicance least signicant
dierences (LSD) were computed at the . probability level
to compare means. All analyses were performed using the
3. Results and Discussion
3.1. Germination. According to analysis of variance, stratied
seeds showed higher germination rates than nonstratied
seeds for each tested GA3dose. ere were no interactions
between the sources of variation used (cold treatments and
GA3treatments). e GA3treatment had an eect on germi-
nation time so a generalized early germination was observed
when and ppm doses were used (Figure ). In these
cases, aer days, most of the seeds were germinated.
Medium germination time (MGT) for these doses was 19.82±
0.475 and 20.34 ± 0.545,respectively.Regardingppm,
a quick germination was also observed, but not as strong
(MGT = 23.77 ± 1.111), and newly germinated seeds were
ob s er ved until the t h day. Fi nally, t h e ppm d ose mea nt
a good nal germination rate, but the number of germinated
seeds increased until the th day at a gradual rate (MGT =
39.16 ± 2.186).
At the end of the days of the germination test, none
of the sterilized water pretreated seeds were germinated.
All GA3treatments yielded signicantly higher germination
Test da y
F : Germination curves of Gentiana lutea var. aurantiaca
seeds using dierent doses of GA3(, , , and ppm) and
percentages compared to the control. e greater the hor-
monal dose used, the greater the percentage of germination
obtained. Seeds pretreated with ppm of GA3resulted in
the highest germination percentage (%), not signicantly
dierent compared to the ppm (%) or ppm (%)
doses, but signicantly dierent to the ppm dose (%)
e results obtained in Petri dishes germination test are
in accordance with those obtained by P´
ıa et al. ,
which verify the high degree of success in the elimination of
seed dormancy using GA3, this eect being greater with high
doses (Figure ). e highest GA3doses ( and ppm)
e Scientic World Journal
Control 50 100 500 1000
F : Germination percentages of Gentiana lutea var. aurantiaca seeds using dierent GA3doses (, , , and ppm) and control
at the end of the test of days of germination. Bars with dierent letters are signicantly dierent (𝑃 = 0.05) according to LSD.
50 100 500 1000
F : Stem length of Gentiana lutea var. aurantiaca seedlings using dierent GA3doses (, , , and ppm) on the th day
aer sowing. Bars with dierent letters are signicantly dierent (𝑃 = 0.05) according to LSD.
provided a quick and grouped germination. e eect of the
GA3dose of ppm was that the germination was slower
during the rst part of the test compared to higher GA3doses,
but MGT and germination were similar for all three doses.
3.2. Seedlings Development. Analysis of variance shows sig-
nicant dierences between GA3treatments. Stem length
was greater when the GA3dose used was higher. e dose
of ppm provided the longest (. mm) stem, being
signicantly dierent from the others, as well as the ppm
dose (. mm). Signicant dierences were not found
between the remaining doses of ppm and ppm, both
providing the shortest stems of all the treatments (.mm
and . mm, resp.) (Figure ).
Depending on the GA3dose, gentian seedlings showed
dierent growth habits (Figure ). H: the aerial part of the
plant is formed by a basal rosette of well-developed leaves
and the stem is very short. H: the aerial part of the plant is
formed by a basal rosette of well-developed leaves; the stem
is formed and strong. H: the leaves are elongated; they are
arranged along a stem formed with short internodes. H:
small leaves are arranged on a very long and weak stem: long
internodes; light yellow-green color. H: poorly developed
and large leaves: liform appearance; long internodes; light
Considering these ve growth habits the number of plants
showing each of the habits was counted according to the
dierent GA3doses (Figure ). e habit shown by the
seedlings at low doses was similar, H being the predominant.
Meanwhile, for high GA3doses, and ppm, the pre-
dominant growth habit was H, and also H was abundant. In
both, ppm and ppm, none of the seedlings showed
the H habit, which is considered the best for the gentian
e negative eect on the development of the seedlings
was observed only at and ppm doses, whereas a
high percentage of seedlings showed o-type growth habits
with long stems. Using these doses, seedlings were very weak,
poorly developed, and with a threadlike appearance, there-
fore, more susceptible to low temperatures, insect attacks,
physical breakage, or drying out. While at low doses ( ppm
seedlings morphology was very similar to that observed in
nature, where the seedlings are formed by a basal rosette, like
other perennial mountainous plants, protecting themselves
from the hard conditions of the mountain climate.
e best GA3pregerminative doses to break seed dormancy
of Gentiana lutea L. var. aurantiaca were , , and
ppm. Doses of and ppm GA3not only allow
obtaining a high and uniform germination, but also produce
high percentages of weak and o-type seedlings. e best
doses to optimize seedling habit were and ppm. is
e Scientic World Journal
F : Growth habits (H–H) of Gentiana lutea var. aurantiaca seedlings on the th day aer sowing treated seeds with GA3(, ,
, and ppm).
50 100 500 1000
F : Percentage of Gentiana lutea var. aurantiaca seedlings
showing dierent growth habits for each of the GA3doses (, ,
, and ppm). Bars with dierent letters for each GA3doses
are signicantly dierent (𝑃 = 0.05) according to LSD.
led to a high germination rate and good seedling morphology,
as the starting point for gentian regular cultivation.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
e authors acknowledge the Regional Ministry of Education
of the Junta de Castilla y Le´
on and the European Social
Fund PIRTU Grants (Orden EDU//), the Regional
Ministry of the Environment of the Junta de Castilla y Le´
Project no. //, the sta from the Forest and
Range Management Research Institute (CRA Agricultural
Research Council-Trento), and Asociaci´
on Promotora de la
Genciana y otras plantas de inter´
es de la monta˜
leonesa for collaborating to collect gentian material.
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