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International Journal of Horticultural Science and Ornamental Plants Vol. 3(2), pp. 059-063, November, 2017. ©

International Journal of Horticultural Science and Ornamental Plants

Vol. 3(2), pp. 059-063, November, 2017. © www.premierpublishers.org. ISSN: XXXX-XXXX

Research Article
Research Article

IJHSOP

Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium longiflorum L.)

Sarra Arbaoui 1 *, Sihem Soufi 2 , Taoufik Bettaieb 3

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

Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium longiflorum L.)

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Arbaoui et al . 060 Figure 1. Lily Scales ( Lilium longiflorum L.) grown in vitro

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/m 2 /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.

Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium longiflorum L.)

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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

2.330.19 c

0,75 0.07 d 0.92 0.07 c 1.22 0.13 b 1.72 0.12 a 1.63 0.08 a

M2

0.5

100

3.51 0.24 5.43 0.20 5.64 0.27 5.42 0.19

b

M3

1

100

a

M4

1.5

100

a

M5

2

100

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 0.18 0.07e 0.17 0.10e 0.28 0.08b 0.26 0.08c 0.24 0.09d 0.27 0.11b 0.310.05a

1.19 0.05f 1.20 0.09f 1.33 0.04e 1.320.08 d 1.41 0.12 c 1.44 0.14c 1.71 0.07b 2.10 0.18 a

MB2

100

MB3

100

MB4

100

MB5

100

MB6

100

MB7

100

MB8

100

MB9

100

0.28 0.07b

1.85 0.10 b

Values followed by different letters are significantly different (Duncan test, 0.01).

letters are significantly different (Duncan test, 0.01). Liquid phase Solid phase Figure 2 . Lilium vitroshoots

Liquid phase

Solid phase

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

Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium longiflorum L.)

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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 7.150.12 a 7.200.09 a 7.010.11 b

1.72 0.12 2.18 0.17 2.22 0.10 2.15 0.15

b

0.310.05 b 0.410.08 a 0.390.07 a 0.420.05 a

2.10 0.18 b 2.420.15 a 2.54 0.09 a 2.600.11 a

MC2

a

MC3

a

MC4

a

Values followed by different letters are significantly different (Duncan test, 0.01).

letters are significantly different (Duncan test, 0.01). Figure 2. Vitroplants of lily ( Lilium longiflorum L.)

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.

Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium longiflorum L.)

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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.

Horticultural Science and Ornamental Plants 3(2): 059-063. Copyright: © 2017. Arbaoui et al . This is

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.

Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium longiflorum L.)