The auxins liability often limits expected effects on in vitro culture of plant tissues. In this regard, β-cyclodextrine was used as protective substance to auxins in order to improve rooting and in vitro bulbing of lily (Lilium longiflorum L.). This oligosaccharide was added at a concentration of 10, 20 and 30 mg/l to the rooting medium containing mineral and vitamin additives of Murashige and Skoog (MS), 1.5 mg/l of indole-3-butyric acid (IBA), 30 g/l of sucrose and 6 g/l of agar. After 30 days of culture, in order to enhance in vitro bulbe formation, a liquid medium supplemented with 6-benzylaminopuine (BAP) was added to form a double phase (solid / liquid) media. The results obtained showed that β-cyclodextrin improves the rooting of lily in vitro shoots. Indeed, the average number of roots increases from 5.64 to 7.20 roots per vitro-plant in the medium supplemented with β-cyclodextrin and root length increased from 1.72 to 2.2 cm compared to control.

1. Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium longiflorum L.)
IJHSOP
Effects of β-cyclodextrin on in vitro rooting and bulbing
of lilium (Lilium longiflorum L.)
Sarra Arbaoui1*, Sihem Soufi2, Taoufik Bettaieb3
1,2,3
Horticultural Science Laboratory, Tunisian National Institute of Agronomic Science, University of Carthage,
Tunisia.
The auxins liability often limits expected effects on in vitro culture of plant tissues. In this regard,
β-cyclodextrine was used as protective substance to auxins in order to improve rooting and in
vitro bulbing of lily (Lilium longiflorum L.). This oligosaccharide was added at a concentration of
10, 20 and 30 mg/l to the rooting medium containing mineral and vitamin additives of Murashige
and Skoog (MS), 1.5 mg/l of indole-3-butyric acid (IBA), 30 g/l of sucrose and 6 g/l of agar. After
30 days of culture, in order to enhance in vitro bulbe formation, a liquid medium supplemented
with 6-benzylaminopuine (BAP) was added to form a double phase (solid / liquid) media. The
results obtained showed that β-cyclodextrin improves the rooting of lily in vitro shoots. Indeed,
the average number of roots increases from 5.64 to 7.20 roots per vitro-plant in the medium
supplemented with β-cyclodextrin and root length increased from 1.72 to 2.2 cm compared to
control.
Keywords: Micropropagation, growth hormones, auxin, lily, β-cyclodextrin
INTRODUCTION
Nowadays, in vitro plant propagation is among widely used
techniques throughout the world. However, the multiple
scientific techniques and protocols developed do not
usually provide a convincing result especially with a
number of recalcitrant species. Improvement of these
techniques and protocols should be undertaken even for a
readily micropropagable species. Several studies have
been carried out focusing on rooting phase that represents
a major handicap to micropropagation procedures of
several species. In the case of lily (Lilium longiflorum L.),
in vitro rooting does not raise problems, although, its
enhancement is necessary for a positively impact on the
acclimatization of vitroplants or in the development of in
vitro bulblet. Several studies have shown that focused
artificial growth hormones, particularly, auxins are
constituted by molecules that are not quite stable and
labile. To overcome this handicap, additives like
cyclodextrin can be used in in vitro culture. These additives
are cage molecules that have the property of including
certain organic molecules such as auxins and protecting
them against photodegradation or oxidation (Tsorteki et
al., 2004). Complexes of cyclodextrin / auxin formed have
no covalent bond and can dissociate easily to release the
two components without their properties being affected.
Hence, β-cyclodextrin can promote auxin’s stability and
allows its slow release in favor of the plant cultured in vitro
(Tsorteki et al., 2004). This investigation aims to study the
effect of this oligosaccharide on rooting and in vitro bulbing
of lily (Lilium longiflorum L.).
MATERIAL AND METHODS
Vitroshoots used for the in vitro rooting experiments of lily
were regenerated by scale culture isolated from young
bulbs of the Amsterdam variety (Figure 1). The shoots
produced are multiplied during five subcultures of four
weeks each before their transplanting on the double-phase
(solid and liquid) rooting and bulbing media (Figure 2).
*Corresponding author: Sarra Arbaoui, Horticultural
Science Laboratory, Tunisian National Institute of
Agronomic Science, University of Carthage, Tunisia.
Email: saraharb@hotmail.fr
International Journal of Horticultural Science and Ornamental Plants
Vol. 3(2), pp. 059-063, November, 2017. © www.premierpublishers.org. ISSN: XXXX-XXXX
Research Article

2. Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium longiflorum L.)
Arbaoui et al. 060
Figure 1. Lily Scales (Lilium longiflorum L.) grown in vitro producing shoots
Table 1. Concentrations of BAP and sucrose supplemented to the liquid
phase media used for bulbing lily (Lilium longiflorum L.)
Culture medium BAP (mg/l) Sucrose (g/l)
MB1 0 30
MB2 0 45
MB3 0 60
MB4 0,5 30
MB5 0,5 45
MB6 0,5 60
MB7 1 30
MB8 1 45
MB9 1 60
Culture media used are composed of the minerals and
vitamins of Murashige and Skoog (MS, 1962)
supplemented with indole-3-butyric acid (IBA),
benzylaminopurin (BAP), sucrose and β-cyclodextrin.
The solid phase of culture media used for rooting vitro-
plants, was supplemented with 30 g/l sucrose, solidified
with 6g/l of agar and poured into 500 ml jars. The
concentrations of IBA used were as follows: M1 = 0 mg/l;
M2 = 0.5 mg/l; M3 = 1 mg/l; M4 = 1.5 mg/l; M5 = 2 mg/l.
The liquid phase of media used for bulbing was
supplemented with BAP and sucrose at different
concentrations (Table 1) and poured, 30 days later, into
the medium M4 that gave the highest root number and
length during the preceding experimentation.
In a second test, different doses (MC1: 0 mg/l, MC2
medium: 10 mg/l, MC3 medium: 20 mg/l, MC4 medium: 30
mg/l) of β-cyclodextrin were added to the solid phase of
rooting medium M4, and to the MB8 bulb medium. These
two media were chosen because they had the best results
in the previous test.
pH of culture media was adjusted to 5.8 before its
sterilization by autoclaving at 120°C under a pressure of 1
bar for 20 minutes. Cultures were carried out under a
photoperiod of 16 hours, a luminous intensity of 36
μmole/m2/s provided by fluorescent tubes (Sylvania Gros
Lux) and a temperature of 23 °C ±1 °C.
Root development was evaluated after 30 days of culture
and bulb development was evaluated after 60 days of
culture on the double-phase media. Observations were
recorded on rooting rate, number of roots per vitroshoot,
average root length, bulbing rate, size of obtained bulbs,
expressed evaluated by weights and circumferences.
STATISTICAL ANALYSIS
The data was analyzed with the help of SAS v.9.1.3
software using GLM III procedure for analysis of variance.
The respective averages were separated by Duncan
multiple range test (1% threshold). Results obtained are
represented as means ± standard deviation.
RESULTS
In vitro rooting experiments of lily (Lilium longiflorum L.) on
culture media supplemented with IBA led to different
results (Table 2). Regardless of auxin concentrations, a
100 % rooting was obtained while 85% rooting occurred in
the absence of auxin. Vitroplant rooting in control medium
is probably explained by a high content of endogenous
auxins and the exogenous application of IBA globally
improves rhizogenesis.
The number of neoformed roots on vitroplants depends on
the culture medium as well. Indeed, the media M3, M4 and
M5 supplemented, respectively, by 1; 1.5 and 2 mg.l-1 of
IBA provided a development of about 5.5 roots per
vitroplant.
In auxin free medium, the number of roots per vitroplant is
2.3. The longest root, exceeding 1.5 cm were obtained on
medium M4 and M5 containing 1.5 and 2 mg.l-1 of IBA.
Roots were shorter on the control medium, hormone free
media, as well as on the M2 and M3 media.

3. Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium longiflorum L.)
Int. J. Hort. Sci. Ornam. Plants 061
Table 2. Effects of IBA on the in vitro rooting of lily (Lilium longiflorum L.)
Culture medium IBA (mg.l-1
) Rooting rate (%) Number of roots per vitropousses Average root length (cm)
M1 0 85 2.33 0.19 c 0,75  0.07 d
M2 0.5 100 3.51  0.24 b 0.92  0.07 c
M3 1 100 5.43  0.20 a 1.22  0.13 b
M4 1.5 100 5.64  0.27 a 1.72  0.12 a
M5 2 100 5.42  0.19 a 1.63  0.08 a
Values followed by different letters are significantly different (Duncan multiple range test, 0.01).
Table 3. Effects of IBA on in vitro bulbing of lily (Liliumlongiflorum L.) after 60 days of culture on ‘double
phase’ medium.
Culture Medium Bulbing (%) Bulbsfreshweight (g) Bulbscircumference (cm)
MB1 100 0.17  0.08e 1.19  0.05f
MB2 100 0.18  0.07e 1.20  0.09f
MB3 100 0.17  0.10e 1.33  0.04e
MB4 100 0.28  0.08b 1.32 0.08 d
MB5 100 0.26  0.08c 1.41  0.12 c
MB6 100 0.24  0.09d 1.44  0.14c
MB7 100 0.27  0.11b 1.71  0.07b
MB8 100 0.31 0.05a 2.10  0.18 a
MB9 100 0.28  0.07b 1.85  0.10 b
Values followed by different letters are significantly different (Duncan test, 0.01).
Figure 2. Lilium vitroshoots in double-phase medium (solid/liquid) for rooting and bulbing
As per the obtained results, it appears that the use of auxin
gave visible effects of improved rooting was improved in
its presence. Previous investigators also obtained
comparable results related to rooting with IBA in Lilium
longiflorum L. (Azadi and Khosh-Khui, 2007 and Matmati
et al., 2014). They showed that in vitro rooting of lilies is
possible in absence of auxins, and its addition contributed
significantly to the enhancement of rooting rate, number
and root length.
The in vitro formation of lily bulbs was observed on all
vitroshoots, but significant differences in weight and size
of bulbs were recorded (Table 3) (Figure 3). The highest
weight and circumference were obtained on the MB8
culture medium supplemented with 1 mg.l-1 of BAP and 45
g.l-1 of sucrose.
This stimulatory effect of in vitro bulb formation of lilies has
been observed by several authors including those of Datu
and Bhojwani (1995), Matmati et al. (2014). BAP free
media allows bulb formation but do not appear to stimulate
their growth.
Also, sucrose concentration affects significantly in vitro
formation and growth of lily bulb since significant
differences in size and fresh weight of bulb were recorded
between the different treatments.
The highest fresh weight and circumference were
obtained with the higher concentrations of sucrose (4.5
and 6%) independent of BAP concentration. However,
sucrose concentration of 3% does not constitute a limiting
factor for bulb formation. Although, the formed bulbs
remain relatively small in size compared to those obtained
on media supplemented with high concentration of
Liquid phase
Solid phase

4. Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium longiflorum L.)
Arbaoui et al. 062
Table 4. Effects of β-cyclodextrine on in vitro rooting and bulbing of lily (Liliumlongiflorum L.)
Culture Medium Number of roots/ in vitro shoot Rootslengh (cm) Bulbs fresh weight (g) Bulbscircumference (cm)
MC1 5.64  0.27c 1.72  0.12 b 0.31 0.05 b 2.10  0.18 b
MC2 7.15 0.12 a 2.18  0.17 a 0.41 0.08 a 2.42 0.15 a
MC3 7.20 0.09 a 2.22  0.10 a 0.39 0.07 a 2.54  0.09 a
MC4 7.01 0.11 b 2.15  0.15 a 0.42 0.05 a 2.60 0.11 a
Values followed by different letters are significantly different (Duncan test, 0.01).
Figure 2. Vitroplants of lily (Lilium longiflorumL.) after bulbing
sucrose. These findings are similar some studies showing
that sucrose concentrations higher than the concentration
usually used in in vitro culture (2 to 3%) enhances the
formation and growth of bulbs in several species with
underground storage organs. Also the, in vitro formation of
tulip bulbs is improved by the addition of 4 to 6% of sucrose
in culture media (Nishiuchi, 1980). Likewise for lily, number
and size of bulbs formed in vitro are highest when sucrose
is used in culture media at a concentration of 9%
(Takayama and Misawa, 1979 quoted by Datu and
Bhojwani, 1995). For potatoes, in vitro tuberization was
favored by 6% sucrose content (Espinosa et al., 1986 and
Bettaieb et al., 2002).
According to the results presented in table 4, the presence
of β-cyclodextrin in culture medium improves vitroshoot
rooting since the mean number and length of roots
obtained are superior to those obtained in the control
medium. Indeed, number of roots increases from 5.64 to
7.20 roots per vitroplant and length changes from 1.72 to
2.2 cm. However, rooting is general even in the absence
of β-cyclodextrin.
The increase in number and length of the roots had
positive repercussions on bulb size; the mean weight
increased in the presence of this additive from 0.31 g to
0.42 g and the circumference from 2.10 to 2.60 cm.
According to the studies of Mura et al. (1995), presence of
β-cyclodextrin in culture medium increases auxin solubility
which positively affects in vitro rooting of olive vitroplants.
Improvement effect of β-cyclodextrin for invitro rooting has
been also reported on jojoba (Apostolo et al., 2001)
artichoke (Brutti et al., 2000) (Dridi, 2003) and in Nolina
recurvata Hemsl (Bettaieb et al., 2008).
According to Bellanger et al. (1992), auxin is an unstable
molecule that is protected by the β-cyclodextrin against
photodegradation or oxidation and allows its slow and
controlled release. Cyclodextrins are molecular cagesable
to include certain molecules and solubilize them by their
cavity, thus forming complexes named inclusion
complexes (Van der veen et al., 2000; Apostolo et al.,
2001). Del Valle, (2004) reported that the main force
causing the formation of complexes is the energy
stabilization of the system by the replacement in the cavity
of water molecules with high enthalpy of hydrophobic
molecules which create apolar-apolar associations.
CONCLUSION
On a double-phase culture medium (solid / liquid),
prepared for rooting and bulb development and growth,
vitroshoots of lilies develop a root hair of 5.64 roots and
1.72 cm long and vitrobulbs of 0.31g and 2.10 cm of
circumference. The double-phase culture medium was
consisted of mineral and vitamin additives of MS (1962),
1.5 mg/l of IBA, 30 g/l of sucrose and 6 g/l of agar in its
solid phase and of a similar medium without agar in which
the IBA was substituted with 1 mg of BA and added with
45 g/l of sucrose.
The addition of an auxin protective oligosaccharide, β-
cyclodextrin, to rooting medium at a concentration of 30
mg/l improves rooting and bulb growth. Vitroplants
obtained have a root hair larger than that obtained from
control medium, composed of 7.20 roots, length of 2.22 cm
and bulbs of 0.39 g circumference of 2.54 cm.

5. Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium longiflorum L.)
Int. J. Hort. Sci. Ornam. Plants 063
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Accepted 20 April, 2017
Citation: Arbaoui S, Soufi S, Bettaieb T (2017). Effects of
β-cyclodextrine on invitro rooting and bulbing of lilium
(Liliumlongiflorum L.). International Journal of Horticultural
Science and Ornamental Plants 3(2): 059-063.
Copyright: © 2017. Arbaoui et al. This is an open-access
article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium,
provided the original author and source are cited.