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Propagation of Ornamental Plants
Vol. 14, № 3, 2014: 133-138

THE EFFECTS OF PLANT GROWTH REGULATORS AND INCUBATION TEMPERATURES ON

GERMINATION AND BULB FORMATION OF FRITILLARIA PERSICA L.

Suleyman Kizil1* and Khalid Mahmood Khawar2

1
Dicle University, Faculty of Agriculture, Department of Field Crops, Ziraat Fakultesi str., 21280 Diyarbakir,
Turkey, *Fax: + 90 41 22488153, *E-mail: suleymankizil@gmail.com
2
Ankara University, Faculty of Agriculture, Department of Field Crops, 1 Kecioren Fatih str.,
06110 Ankara, Turkey

Abstract
Fritillaria persica L. with large attractive flowers is native to Western Asian countries of the Middle East
including Turkey. Wild populations of this species have shown sharp decrease during last few decades due
to habitat destruction for number of socio-economical and anthropological reasons. F. persica is propagated
through bulbs and seeds. The seeds have significantly high dormancy such that few seeds germinate under
natural conditions. Therefore, this study developed a seed dormancy break protocol using MS medium contain-
ing variants of BAP with and without IBA incubated at 4°C in dark. Maximum seed dormancy break (80.00 ±
0.14%) was registered on MS medium enriched with 2.0 mg l-1 BAP plus 1.0 mg l-1 IBA and maximum bulblet
induction (40.0 0 ± 0.71) was noted on MS medium containing 1.0 mg l-1 BAP plus 1.0 mg l-1 IBA. Similarly,
alternating incubation temperatures of 4° and 10°C for variable durations in days influenced seed germination
and bulblet induction variably with 100% seed germination and bulblet induction at 75 days incubation at 4°C
followed by 15 days incubation at 10°C. The results also suggested that minimum incubation period of 30
days at 4°C followed by incubation at 10°C for 60 days was required to break seed dormancy. The increase in
bulblet diameter was achieved on MS medium containing 50 mg l-1 sucrose by incubating the bulblets at 4°C
for 30 days. Rooting of the Fritillaria bulblets was obtained on MS medium enriched with 0.5 mg l-1 NAA. This
propagation method could be exploited practically avoiding any seasonal constraints to obtain plant material
and suggests a positive step further for in vitro propagation of F. persica.

Key words: bulblets, dormancy, Persian lily, PGR(s), seed propagation

INTRODUCTION the immediate surroundings. All of them also agree that

Genus Fritillaria (Liliaceae) includes more than
100 species that are widely distributed in temperate
climates of South European and West Asian countries
of the Middle East (De Hertogh and Le Nard 1993)
including Turkey (Bryan 2002, Tubives 2013). They
flower during spring and their use as border plants in
gardens (De Herthogh and Le Nard 1993) and as cut
flower for interior decorations is well established. Al-
though species are captivating plants, their propagation
through bulbs, bulb scales or by seeds is quite difficult
and limited.
Although in vitro multiplication of Fritillarias have
been reported by Sun et al. (1977), Kukulezanka et al.
(1989), Gao et al. (1999), Paek (1996), Paek and Murthy
(2002), and Mohammadi-Dehcheshmeh et al. (2007),
all these authors agree that in vitro regenerated bulbs
are difficult to establish under external conditions as
the bulbs are sufficiently weak to resist abiotic stress in
bulbs obtained from natural sources are generally highly
contaminated with bacteria and fungi, which prevents
successful use of bulb scales in in vitro propagation
studies. Moreover, use of bulb scales as explant could
also have negative effect on their natural populations
(Witomska and Lukazewska 1997).
Each plant bear number of capsules containing a
few to more than 1800 seeds each. F. persica seeds
usually have underdeveloped embryos, with poor seed
germination. The seedlings are very weak and have
low survival rates during early periods of growth due
to number of biotic and abiotic stresses. Germinated
seedlings grow very slow and the bulbs mature in 4-6
years (De Hertogh and Le Nard 1993). Natural popula-
tions of Fritillaria persica need conservation through
alternative methods employing fast vegetative or seed
propagation techniques (Ulug et al. 2010) that can
guarantee commercial scale production. This could

Received: June 24, 2014 Accepted: August 29, 2014

133
Suleyman Kizil and Khalid Mahmood Khawar. In vitro germination and bulblet formation of Fritillaria persica
help providing of sufficient material to propagate plants
commercially and reinforce wild populations as well.
This study aimed to accelerate seed germination
of F. persica by checking the effects of BAP plus IBA
and duration of alternating incubation temperatures of
4°C and 10°C on seed dormancy release without losing
stable morphological features of the newly germinat-
ing plants.
MATERIALS AND METHODS
Plant material and experiments
F. persica mature seed capsules were collected from
the plants growing at the experimental fields of the De-
partment of Field Crops, Dicle University, Diyarbakir,
Turkey. These were stored in dark at room temperature
(24 ± 1°C) for one year. After extraction from the dried
capsules (Fig. 1A) seeds were surface disinfected using
100% commercial bleach (Ace, Turkey containing 5%
NaOCl) for 15 min. Subsequently, they were rinsed
3 × 3 min with sterile distilled water. The surface
disinfected seeds were cultured on 0.65% (w/v) agar
(Duchefa, Harlenm, The Netherlands) solidified MS
(Murashige and Skoog 1962) medium containing 0.5,
1.0, 1.5, 2.0, 2.5, and 3.0 Gibberellic acid (GA3) at 4°C
or 1.0 mg l-1 6-benzylaminopurine (BAP) plus 0.0,
1.0, and 2.0 mg l-1 indole-3-butyric acid (IBA; three
combinations) or 2.0 mg l-1 BAP plus 0.0, 1.0, and 2.0
mg l-1 IBA (three combinations) for germination at 4 ±
1°C in dark for 90 days.
In another experiment, the seeds were incubated on
0.65% (w/v) agar-solidified MS medium at alternating
temperatures of 4°C followed by incubation at 10°C and
vice versa for 0 and 90 days, 15 and 75 days, 30 and 60
days, 45 and 45 days, 60 and 30 days, 75 and 15 days
and 90 and 0 days, respectively, for each temperature
range in dark.
The germinated seeds were incubated on 0.65%
(w/v) agar-solidified MS medium at 24 ± 1°C for bulblet
induction under cool white fluorescent light (42 µM m-2
s-1) of 16 h light photoperiod for 15-20 days for in vitro
bulblet induction.
Thereafter, they were separated and subcultured
on 0.65% (w/v) agar-solidified MS medium con-
taining 50 g l-1 sucrose to increase bulblet diameter.
Developing bulblets were rooted on 0.65% (w/v) agar-
solidified MS medium supplemented with 0.5 mg l-1
α-naphthaleneacetic acid (NAA) at 24 ± 1°C under 16
h light photoperiod provided by cool white fluorescent
light (42 µM m-2 s-1).
The pH of all variants of the medium was adjusted to
5.6-5.8 with 1 M KOH or 1 M HCl before autoclaving
at 121°C and 118 kPa for 20 min.
Observations and statistical analysis
Each experimental treatment contained 400 seeds
134
divided into 80 replicates containing five explants each.
Data were analysed by one-way ANOVA using software
SPSS 18.0. The significance of differences among
means was determined using Duncan’s Multiple Range
test. All percentage data were square-root transformed
(Snedecor and Cochran 1989) before statistical analysis.
RESULTS
No F. persica seed germination was recorded on
any concentration of GA3 at 4°C in dark.
Effects of BAP and IBA on seed germination at 4°C
Varying concentration of BAP and IBA affected
seed germination differently. The seed germination
ranged from 46.67 ± 0.65% to 80.00 ± 0.14%. The best
result was registered on MS medium containing 2.0 mg
l-1 BAP plus 1.0 mg l-1 IBA. The seed germination or
emergence of embryos started at 89th-95th day after in-
cubation and seeds germinated with variable emergence
of radicles. Germination completed in next 5-8 days.
Therefore, 89th day of incubation could be treated as
benchmark for minimum period to break seed dormancy
irrespective of the type of seed treatment (Table 1).
All germinating seeds did not induce bulblets.
Strong and vigorous seeds induced bulblets on tips
of roots after 118 to 129 day of incubation. First and
maximum bulblet induction of 40.00 ± 0.71% was noted
on 1.0 mg l-1 BAP plus 1.0 mg l-1 NAA after 118 days
of culture (Table 1).
Bulblet induction rate varied with range of 13.30 ±
0.52 to 40.00 ± 0.71%. Leaf length had range of 5.67
± 0.46 to 14.3 ± 0.54 cm with longest leaves on MS
medium containing 2 mg l-1 BAP + 2 mg l-1 IBA.
Number of bulblets per germinated seed varied
from 0.70 ± 0.01 to 1.00 ± 0.00 (Table 1). The maxi-
mum number of bulblets was noted on MS medium
containing 1.0 mg l-1 BAP or 1.0 mg l-1 BAP plus 1.0
mg l-1 IBA. Minimum number of 0.70 ± 0.01 bulblets
per germinated seed were noted on 1.0 mg l-1 BAP plus
2.0 mg l-1 IBA (Table 1).
The bulblet diameter varied and had range of 0.23
± 0.04 cm to 0.42 ± 0.02 cm. The seed weight was not
homologue to number of bulblets per germinated seed
and registered maximum gain in weight (0.44 ± 0.01
g) on MS medium containing 2.0 mg l-1 BAP plus 2.0
mg l-1 IBA (Table 1).
Effects of alternating 4°C and 10°C incubation tem-
peratures on seed germination and bulblet formation
No bulblet induction was noted on 4°C incubation
for 0 and 15 days followed by incubation at + 10°C for
90 and 75 days respectively. Variable seed germination
was recorded on other alternating incubation tempera-
tures. Protrusion of radicles or seed germination started
at on 88-95th day after incubation. Irrespective of the
 Propagation of Ornamental Plants
Vol. 14, № 3, 2014: 133-138

Table 1. Effects of BAP and IBA on seed germination and number bulblet induction.

Number of Number
Bulblet Number of Bulblet Bulblet
BAP IBA Germination days to of days Leaf length
induction bulblets per diameter weight
(mg l-1) (mg l-1) (%) first seed to induce (cm)
(%) seed (cm) (g)
germination bulblets
1.0 0.0 46.67 ± 0.65 b 90.00 26.67 ± 0.63 b 119.00 1.00 ± 0.00 5.67 ± 0.46 f 0.24 ± 0.02 b 0.31 ± 0.01 b
1.0 1.0 66.67 ± 0.44 ab 89.00 40.00 ± 0.71 a 118.00 1.00 ± 0.00 8.22 ± 0.15 e 0.29 ± 0.01 b 0.35 ± 0.01 b
1.0 2.0 80.00 ± 0.38 a 89.00 13.30 ± 0.43 c 119.00 0.70 ± 0.01 9.31 ± 0.36 d 0.26 ± 0.03 b 0.41 ± 0.02 a
2.0 0.0 80.00 ± 0.89 a 94.00 13.30 ± 0.26 c 126.00 0.71 ± 0.03 11.35 ± 0.33 c 0.27 ± 0.01 b 0.35 ± 0.01 b
2.0 1.0 80.00 ± 0.14 a 94.00 13.30 ± 0.13 c 129.00 0.71 ± 0.01 12.94 ± 0.42 b 0.42 ± 0.02 a 0.46 ± 0.02 a
2.0 2.0 66.67 ± 0.25 ab 95.00 13.30 ± 0.52 c 120.00 0.73 ± 0.02 14.33 ± 0.54 a 0.23 ± 0.04 b 0.44 ± 0.01 a
Mean values ± standard error within a column followed by the same letter are not significantly different according Duncan’s
multiple range test at p ≤ 0.05.

alternating incubation period from 4°C to 10°C, 88
days of incubation was the minimum time to protrude
radicles from the seeds (Table 2).
When excluding seeds on non-germinating treat-
ments, seed germination percentage and bulblet induc-
tion percentage ranged from 93.33 ± 0.82 to 100.00 ±
0.00 and from 93.33 ± 0.91 to 100.00 ± 0.00%. Maxi-
mum (100%) seed germination was noted on 4 different
alternating incubation treatments [45 days incubation at
4°C and 45 days incubation at 10°C (Fig. 1B), 60 days
incubation at 4°C and 30 days incubation at 10°C, 75
days incubation at 4°C and 15 days incubation at 10°C
and 90 days incubation at 4°C and 0 days incubation
at 10°C] (Table 2).
Vigorously grown seeds showed bulblet induc-
tion on root tips after 116 to 126 days of incubation.
Maximum (100.00%) bulblet induction was noted on
incubation of 75 days at 4°C followed by alternating
temperature of 10°C for 15 days or 90 days incubation
at 4°C (Table 2).
Maximum number of one bulblet per germinating
seed and first bulblet induction was noted on 75 day
incubation at 4°C followed by 15 days incubation at
10°C (Fig. 1C) after 116 days of incubation (Table 2).
The leaf length on these newly formed bulblets
ranged 7.94 ± 0.86 to 16.67 ± 0.98 cm with maximum
increase in length when the leaves were incubated at
4°C for 90 days (Table 2). Bulblet diameter on the
germinated bulblets registered statistical similarities
with maximum gain (0.53 ± 0.02 cm) on alternating
45 day 4°C, 45 day 10°C incubation temperature treat-
ments (Table 2).
Maximum bulblet weight gain (0.67 ± 0.02 mg)
was noted on alternating 75 day 4°C and 15 day 10°C
incubation treatments. These bulblets induced increased
diameter on MS medium containing 50 g l-1 sucrose to
increase bulblet diameter (Fig. 1D).
Effects of alternating 10°C and 4°C incubation tem-
peratures on seed germination and bulblet formation
Seed germination (100.00 ± 0.00%) was noted on
two treatments only, when the seeds were incubated at
4°C for 90 days or the seeds were incubated at 10°C
for 15 days followed by incubation at 4°C for 75 days.
Rest of the incubation temperature treatments failed to
induce any seed germination. First seed germination
treatment was noted after 90 days of culture and first
bulblet induction was noted after 120 days of culture
(Table 3). Both showed variable number of bulblets per
seed, leaf length, bulblet diameter, and bulblet weight.

Table 2. Effects of alternating 4°C and 10°C temperatures on seed germination and bulblet induction.

Incubation condi- Number of

Number of days Bulblet Number of Leaf Bulblet Bulbet
tions (days) Germination days to
to first seed induction bulblets per length diameter weight
(%) induce
germination (%) seed (cm) (cm) (g)
+4°C +10°C bulblets
0 90 0.00 ± 0.00 b 0.00 0.00 ± 0.00 c 0.00 0.00 ± 0.00 b 0.00 ± 0.00 d 0.00 ± 0.00 0.00 ± 0.00 c
15 75 0.00 ± 0.00 b 0.00 0.00 ± 0.00 c 0.00 0.00 ± 0.00 b 0.00 ± 0.00 d 0.00 ± 0.00 0.00 ± 0.00 c
30 60 93.33 ± 0.82 a 88.00 93.33 ± 0.91 a 118.00 0.72 ± 0.03 a 10.91 ± 0.17 b 0.41 ± 0.01 0.42 ± 0.01 b
45 45 100.00 ± 0.00 a 90.00 66.67 ± 0.67 b 118.00 0.72 ± 0.09 a 11.11 ± 0.94 b 0.53 ± 0.02 0.41 ± 0.01 b
60 30 100.00 ± 0.00 a 91.00 66.67 ± 0.29 b 119.00 0.74 ± 0.02 a 7.94 ± 0.86 c 0.51 ± 0.02 0.33 ± 0.01 b
75 15 100.00 ± 0.00 a 94.00 100.00 ± 0.00 a 116.00 1.00 ± 0.01 a 15.63 ± 0.57 a 0.51 ± 0.02 0.67 ± 0.02 a
90 0 100.00 ± 0.00 a 95.00 100.00 ± 0.00 a 126.00 0.91 ± 0.05 a 16.67 ± 0.98 a 0.42 ± 0.06 0.44 ± 0.01 b

Mean values ± standard error within a column followed by the same letter are not significantly different according Duncan’s
multiple range test at p ≤ 0.05.

135
Suleyman Kizil and Khalid Mahmood Khawar. In vitro germination and bulblet formation of Fritillaria persica

10
A B

C D
Fig. 1. Fritillaria persica seed germination under in vitro conditions. A) Seeds, B) Germinating seeds on MS medium at 4°C
incubation for 45 days followed by incubation at 10°C for 45 days, C) Bulblet formation on germinated seeds on MS medium
after 75 days incubation at 4°C followed by 15 days incubation at 10°C, D) Bulblets enlargement in MS medium at 24 ± 1°C.
Bar of Fig. 1A = 1.2 cm, 1B = 2 cm, 1C,D = 1.3 cm

DISCUSSION embryos that display morpho-physiological dormancy

Seed dormancy delays seed germination until appro-
priate time to avoid unfavourable or harmful growing
conditions (Baskin and Baskin 2001). Physiological
dormancy prevents protrusion of radicles and elonga-
tion of axis (Baskin and Baskin 2004). Irrespective
of development of differentiating or undifferentiating
tissues, the morphological dormancy results in devel-
opment of underdeveloped embryos. As the embryo
develops it expands away from the base resulting in
emergence that starts several weeks after root growth.
Optimum temperature for post-dispersal embryo devel-
opment and germination is thought to be 4-5°C. Seed-
ling emergence in F. persica occurs during February-
July. It varies every year depending on the temperature
and humidity conditions in the environment.
Fritillaria species have linear, underdeveloped
in line with Baskin and Baskin (2001, 2004), Baskin
et al. (2000), and Finch-Savage and Leubner-Metzger
(2006). Seeds under influence of morphophysiological
dormancy need either treatment with GA3, abscicic acid
(ABA), cytokinins, and auxins (Seo et al. 2011) or a
combination of warm stratification and moist-chilling
to break dormancy (Hilhorst 2011) that results in fast
multiplication and growth of cells followed by pro-
trusion of radicles out of seeds. Therefore, the study
compared both effectiveness of GA3, BAP-IBA and
alternating temperature treatments to ensure the best
conditions for release of seed dormancy.
The results of this study provide systematic infor-
mation about effects of abiotic factors that could help
to release seed dormancy independently of the season.
The study suggested that variable concentrations of

136
 Propagation of Ornamental Plants
Vol. 14, № 3, 2014: 133-138

Table 3. Effects of alternating 10°C and 4°C temperatures on seed germination and bulblet induction.

Incubation con- Number Bulblet Number

ditions (days) induction Number of Bulblet Bulblet
Germination of days to of days Leaf length
(%) bulblets per diameter weight
(%) first seed to induce (cm)
seed (cm) (g)
+10°C +4°C germination bulblets

0 90 100.00 ± 0.00 a 95.00 100.00 ± 0.00 a 126.00 0.91 ± 0.02 a 16.74 ± 0.26 a 0.41 ± 0.01 a 0.42 ± 0.01 a
15 75 100.00 ± 0.00 a 90.00 100.00 ± 0.00 a 120.00 0.42 ± 0.01 b 15.12 ± 0.91 b 0.32 ± 0.01 b 0.24 ± 0.01 b
30 60 0.00 ± 0.00 b 0.00 0.00 ± 0.00 b 0.00 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c
45 45 0.00 ± 0.00 b 0.00 0.00 ± 0.00 b 0.00 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c
60 30 0.00 ± 0.00 b 0.00 0.00 ± 0.00 b 0.00 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c
75 15 0.00 ± 0.00 b 0.00 0.00 ± 0.00 b 0.00 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c
90 0 0.00 ± 0.00 b 0.00 0.00 ± 0.00 b 0.00 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c
Mean values ± standard error within a column followed by the same letter are not significantly different according Duncan’s
multiple range test at p ≤ 0.05.

GA3 were not suitable for seed dormancy break. Gib-
berellic acid is frequently used to induce seed germi-
nation that circumvent requirement for use of other
dormancy breaking compounds in general. However,
deeply (physiologically) dormant seeds fail to respond
to GA3 treatments (Baskin and Baskin 2001). GA3 did
not promote seed dormancy release suggesting factors
other than GA3-ABA balance were responsible for seed
dormancy. The results are further supported by previous
findings by Carasso et al. (2011), who suggested that
gibberellic acid (GA3) did not promote embryo germi-
nation in F. tubiformis subsp. moggridgei.
BAP-IBA treatments and alternating temperature
incubations were helpful to release arrested growth of
embryos only after seed imbibition, when seeds were
cultured on semi-solid agar containing MS medium on
any variant of BAP-IBA. This suggested that different
levels of BAP-IBA had variable interaction with seeds
and each variant of BAP-IBA influenced interactions
between seed morphology, physiology, and biochemical
functions independently and differently by determining
own paths to break seed dormancy in particular man-
ner. The result show partial agreement with Sumlu et
al. (2010), who suggested treatment of stored seeds of
water lily (Nymphaea alba L.) with TDZ under in vitro
conditions to break seed dormancy.
The seeds germination was observed when 4°C
incubation temperature was maintained for minimum of
30 days followed by incubation at 10°C for 60 days that
resulted in increase of cell multiplication and growth
of embryos resulting in seed germination.
Various germination percentages were recorded at
each alternating temperature incubation. The start of
germination was not delayed beyond 95 days of culture
and bulblet induction beyond 129 days in any treatment.
Strong and vigorous seeds induced bulblet formation
on tips of roots after 118 days of incubation.
The second experiment clearly demonstrates impor-
tance of initial temperature during seed germination that
affects plant growth and development. It shows how
initial temperature treatment affects seed dormancy
associated with significant changes in germination be-
haviour. No seed germination was noted when the seeds
were incubated in dark at 10°C followed by incubation
at 4°C for 30 and 60 days, 45 and 45 days, 60 and 30
days, 75 and 15 days, and 90 and 0 days, respectively.
Seed germination only occurred when the seeds were
incubated at 10°C followed by incubation 4°C for 0-90
and 15-75 days. Longer periods of incubation (in days)
at 10°C were inhibitory resulting in arrest of growth
of embryos
The results of these two alternating temperature
range experiments suggest that initial temperature
requirement was very essential element for seed
dormancy break. The results are in partial agreement
with Gao et al. (1997), who suggested that 50 days at
8-10°C followed by 80 days incubation at 3-5°C were
the most important period in the whole process of
relieving seed dormancy of F. thunbergii. Carasso et
al. (2011) found that optimum temperature for embryo
development and germination is thought to be 4-5°C
in F. tubiformis subsp. moggridgei. They found that
prematurely transferring seeds from winter to spring
temperatures (5/10°C) also slowed the progress of
germination. They concluded that in situ seed germi-
nation may occur during the winter under snow cover
or at the end of winter to coincide with snow melt and
warming temperatures. The results of present study
are not in agreement with Mancuso et al. (2012) who
found that F. montana shows a physiological, nondeep
dormancy. Mancuso et al. (2012) emphasise that fresh
seeds are capable of germinating at 5°C during cold
stratification. However, low germination is observed
in dried and refrigerator stored seeds.
Considering the rarity of F. persica and its low ger-
mination, the results of this study suggest that irrespec-
tive of age and the season the seeds can be germinated
under in vitro conditions with effective seed dormancy
break. This protocol can help to grow nurseries for field
culture of the F. persica and provide an important start-

137
 Suleyman Kizil and Khalid Mahmood Khawar. In vitro germination and bulblet formation of Fritillaria persica
ing point for in vitro propagation of this ornamentally
important plant and will contribute positively to ex situ
conservation programmes.
Acknowledgements: This work was supported by
a grant (Project number: 110 O 703) from the Scientific
and Technical Research Council of Turkey (TUBITAK).
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