IJMAP 3 1 6 Paulownia Tomentosa

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    Int. J. Med. Arom. Plants, ISSN 22494340RESEARCH ARTICLE

    Vol. 3, No. 1, pp. 40-46, March 2013

    *Corresponding author: (E-mail) nbenbahri yahoo.fr http://www.openaccessscience.com

    2013 Copyright by the Authors, licensee Open Access Science Research Publisher. [email protected]

    This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC-ND 3.0) License (http://creativecommons.org/licenses/by-nc-nd/3.0)

    Morphological effects of zinc and its accumulation in micropropagated

    plants ofPaulownia tomentosa (Thunb.) Steud.

    Nada BEN BAHRI*, Bochra LARIBI, Taoufik BETTAIEB

    Dpartement dAgronomie et de Biotechnologies Vgtales, Institut National Agronomique de Tunisie. 43, Av.

    Charles Nicolle-1082, Tunis, Tunisie

    *Corresponding author, Tel: +216-71 28 71 10, Fax: +216-71 79 93 91

    Article History: Received 8th

    August 2012, Revised 20th

    January 2013, Accepted 21st

    January 2013.

    Abstract:In vitro essay ofPaulownia tomentosa (Thunb.) Steud. was undertaken to determine the capacity of this me-

    dicinal plant to accumulate heavy metal in its tissues. In this case, the nodal explants were placed on Murashige and

    Skoogs medium (1962), added with 1.0 mg l

    -1

    indole-3-butyric acid (IBA). The ZnSO4 was supplemented to the culturemedium at various concentrations 200, 400, 600, 800 and 1000 M. The MS medium free of Zn was served as control.

    The height of plantlets, the number of nodes, the number and length of roots and the biomass of the in vitro plants were

    determined. The Zn content in plant tissues was measured by an inductively coupled plasma-optical emission spectrome-

    ter (ICP-OES, OPTIMA 2000 DV, PerkinElmer, USA). Results showed that highest Zn level (1000 M) induced a sig-

    nificant reduction in shoot height, (18.86%) and root number (84.9%) of plantlets in comparison to the control. The high-est Zn concentration in the culture medium caused also an important decrease in fresh and dry biomass by 22.8% and

    50.5%, respectively compared to the control. Furthermore, a high Zn accumulation in the plantlet tissues which varied

    from 121g/g dry matter (DM) in the control to 2580 g/g (DM) in the presence of 1000 M Zn was noticed. Conse-quently, Paulownia tomentosa could be considered as a bio-indicator and Zn accumulator medicinal plant.

    Keywords: Micropropagation, Paulownia tomentosa (Thunb.) Steud., medicinal plant, Zn accumulator.

    Abbreviations: IBA: indole-3-butyric acid; MS: Murashige and Skoog medium; Zn: Zinc; DM: Dry Matter.

    Introduction

    Paulownia tomentosa (Thunb.) Steud. is a

    perennial tree species which belonging to theScrophulariaceae family. It is native to centraland western China but its widely distributed inKorea, Japan, and China (Jiang et al. 2004).

    Parts of the plant P. tomentosa (leaves,

    wood, and fruits) have been used in traditional

    Chinese herbal medicine for the treatment of

    tonsillitis, bronchitis, asthmatic attack, and bac-terial infections such as enteritis or dysentery. In

    fact, the flower is the most important material

    used in folk medicine herbs (Kang et al. 1999,Jiang et al. 2004, mejkal et al. 2007). Howev-er, extracts ofP. tomentosa contained many bi-

    oactive compounds such as flavonoids and par-

    ticularly Apigenin. This last has been found to

    show a variety of pharmacological activities,

    including hypotensive (Loizzo et al 2007), anti-

    inflammatory (Gerritsen et al. 1995, Ko et al.

    2004), antispasmotic (Capasso et al 1991), anti-

    oxidant (Cos et al 1998) and vasorelaxant

    (Zhang et al 2000). Besides, Apigenin may exert

    its anti-tumorigenic effect in vivo not only via

    the inhibition of tumor cell proliferation, but

    also via the impairment of the invasive potential

    of tumor cells (Czyz et al. 2005).

    Recently, P. tomentosa has received an in-

    creased attention due to its marketable value for

    wood and biofuel production thanks to its rapidgrowth, high biomass production, and high

    stress tolerance. In fact, this species has exhibit-

    ed strong transpiration rates and real tolerance

    to high metal concentrations in both hydroponic

    and field studies (Liu et al. 2007, Doumett et al.

    2010). Therefore, the aim of the present study

    was to determine for the first time the effects of

    increasing Zinc concentrations on the Paulow-

    nia tomentosa performance, grown in vitro con-

    ditions and the Zn accumulation by plantlets.

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    Int. J. Med. Arom. Plants Effects of zinc in micropropagated plants of Paulownia tomentosa

    Ben Bahri et al.http://www.openaccessscience.com

    [email protected]

    The results will be important to indicate the po-

    tential application of Paulownia tomentosa in

    phytoremediation of contaminated sites.

    Material and methods

    Plants materialNodal explants were collected from old

    plants of Paulownia tomentosa. These explants

    were grown and maintained in the greenhouse

    of the Department of Agronomy and Vegetal

    Biotechnology at the National Agronomic Insti-

    tute of Tunisia.

    Surface disinfection, sterilization and culture

    media

    Disinfection of nodal explants was carried

    out in the laminar airflow chamber by using

    ethanol (few seconds) and soaked in 0.1% (w/v)

    HgCl2 for 5 min. Then, the shoot tips were

    rinsed with sterile distilled water and surface

    sterilized with 10% (w/v) sodium hypochlorite

    for 5 min. Thus, sterilized nodal explants werecultured on Murashige and Skoogs medium(1962) supplemented with 1 mg l

    -1IBA and 3%

    (w/v) sucrose. The medium was gelled with

    0.6% (w/v) agar (Sigma) and the pH was ad-justed to 5.8 with 0.1 N NaOH or HCl before

    autoclaving at 120C for 20 min under a pres-

    sure of 1.1 kg cm-.

    Zn treatments and experimental design

    The metal salt Zinc Sulphate 7-hydrate

    (ZnSO4.7H2O) was added to the culture medium

    at various concentrations: 200, 400, 600, 800

    and 1000 M. The MS medium without adding

    of Zn was served as control. The experimentaldesign was conducted as a factorial randomized

    block with three replications.

    Growth conditions

    The cultures were incubated at 23 1C un-

    der 16/8h (light/dark cycle) photoperiod and ir-

    radiance (36 mol m-2

    s-1

    ) provided by cool-

    white fluorescent lamps.

    Observations were made after four weeks ofculture and focused on plantlet heights, number

    of nodes, root number and average length, bio-

    mass accumulation and the content of dry matter

    Zn.

    Zinc analysis

    Vitro plants were carefully washed with dis-

    tilled water, oven dried at 65oC for about 48

    hours to stop enzymatic reactions and to obtain

    a constant weight, then ground to a fine powder

    before analysis.

    The zinc content was determined after di-

    gesting the plant tissues in a mixture of concen-

    trated HNO3 and H2O2 (2:1 v/v., Sigma-Aldrich,

    Italy) using a digester (VELP Scientifica, Italy).

    After digestion, the Zn content was measured by

    an Inductively Coupled Plasma-optical emissionspectrometer (ICP-OES, OPTIMA 2000 DV,

    PerkinElmer, USA). The ICP-OES analytical

    standard (AA/ICP calibration/check standards

    for environmental analysis, 1 g l-1

    ) for Zn was

    supplied by Sigma-Aldrich (Italy).

    Statistical analysis

    Fifteen explants were used per treatment in

    triplicates. Data were subjected to statisticalanalysis using the program package SAS (SAS

    1999). The one-way analysis of variance(ANOVA) followed by Duncans multiple rangetest at the significance level of 5% was used to

    compare means.

    Results and Discussion

    Zn effect on shoot height and nodes number of

    plants

    The in vitro shoot height of Paulowniatomentosa in the culture media supplemented

    with Zn at different concentrations showed a

    significantly difference between the treatments

    (Table 1).

    The highest shoots were obtained on MS

    culture medium free of Zn (control) and no sig-

    nificant difference was observed in media with200 M of Zn. However, the plant height wassignificantly reduced by 18.86%, compared to

    the control, at 1000 M of Zn.

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    Int. J. Med. Arom. Plants Effects of zinc in micropropagated plants of Paulownia tomentosa

    Ben Bahri et al.http://www.openaccessscience.com

    [email protected]

    When the Zn concentration was up to 400

    M, the number of nodes decreased slightly but

    without a significant difference between treat-

    ments. However, no difference was observed

    between shoots grown on the control or on me-

    dia supplemented with 200 and 400 M of Zn.

    The number of nodes was significantly reducedby 20.63% compared to the control at the high-

    est level of Zn (1000 M) in the culture medium

    (Table 1). Indeed, the presence of Zn in the cul-

    ture media caused a decrease in the aerial parts

    growth of plantlets. These findings corroborate

    those of Gerard et al. (2000) who observed a

    decrease of the biomass produced in cultures of

    Lolium perenne in soils contaminated by Zn. In

    the same context, Wang et al. (2010) showed

    that the germination rate and shoot length of

    Paulownia fortunei decreased when Zn concen-

    trations are higher at mining sites rich in this

    metal. On the other hand, Ruscinska et al.

    (1999) showed that the presence of metals cancause a decrease in biomass, an inhibition of

    photosynthesis, respiration disturbances and de-

    generation of main cell organelles. InBrassica

    napus, Ben Ghnaya (2008) noted that the treat-

    ment in the greenhouse at 2000 M Zn caused a

    decrease reaching 75% in shoot and root bio-

    mass which is depending on the genotype.

    Table 1. Effects of ZnSO4 on vegetative growth and rooting of in vitro plantlets Paulownia

    tomentosa (Thunb) Steud.

    Mean values within the column followed by the same letter in superscript are not significantly differ-

    ent at P

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    Int. J. Med. Arom. Plants Effects of zinc in micropropagated plants of Paulownia tomentosa

    Ben Bahri et al.http://www.openaccessscience.com

    [email protected]

    plants grown on a culture medium supplemented

    with 1000 M of Zn.

    Furthermore, root length average varied sig-

    nificantly in all the treatments (Table 1). Indeed,

    the length of roots was significantly reduced by

    65.8% compared to the control at 1000 M ofZn in the culture medium.

    The effect of Zn on root quality was studied

    in other plants. Schwartz et al. (1999) reported

    that in Thlapsi caerulescens, short roots ar-

    ranged in aggregates in presence of high con-

    centrations of this metal in soil and in uncon-

    taminated soil the roots of the same plant are

    very thin and long.

    In addition, Shi et al. (2011) showed that

    root length, root surface and root volume ofGlochidion puberum grown in lead/zinc (Pb/Zn)

    mine tailings areas were reduced by 70, 80, and

    86%, respectively, compared to that grown in

    uncontaminated soil.

    Effect of Zinc on fresh and dry biomass

    As shown in Figure 2, the addition of Zn to

    the culture medium at different concentrations

    affected severely the fresh and dry biomass ofin

    vitro plantlets and this effect is more pro-

    nounced with increasing levels of Zn.

    Indeed, the fresh biomass decreased from

    9.62 g to 7.42 g in the control and with 1000

    M Zn concentration, respectively. However,

    the fresh biomass of the plantlets treated with

    200 and 400 M of Zn is relatively stable (9 g)

    (Figure 2).

    0

    1

    2

    3

    4

    5

    6

    7

    8

    9

    10

    Biomass(g

    0 200 400 600 800 1000

    Zn concentration (M)

    FW (g) DW (g)

    Figure 2: Effects of ZnSO4 on the biomass ofin vitro plantlets Paulownia tomentosa (Thunb) Steud.

    FW: Fresh weight; DW: Dry weight.

    As well, fresh biomass of plantlets treated

    with 600, 800 and 1000 M Zn is near to 7.5 g.

    A regression by 22.8% and 50.5% on the fresh

    and dry biomass under 1000 M Zn concentra-

    tion compared to the control respectively was

    recorded. These results agree with those found

    by Ben Ghnaya (2008) which observed a signif-

    icant decrease in dry matter weight and fresh

    tissue culture in all the variety ofBrassica

    napus L. Drakkar by adding 500 and 1000 Mof Zn to the culture medium. However, Mench

    et al. (1981) showed that the vegetative growthof maize seedlings is related with the Zn con-

    centration in the solution. On the other hand, the

    addition of different concentrations of Zn to a

    culture of Solanum nigrum seeds (0, 100, 500

    and 1000 mg Zn/kg of sand) affects the growth

    of seedlings. Plants grown at 100 mg kg-1

    showed significantly lower leaf, stem, root and

    total biomass than control ones and plants grow-

    ing at 500 and 1000 mg kg-1

    presented signifi-

    cantly the lowest biomass (Marques et al. 2006).

    In fact, the abiotic stress caused by heavy

    metals, modifies the structure of essential en-

    zyme by altering the protein structure and caus-ing deficiency symptoms in plants. The plasma

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    Int. J. Med. Arom. Plants Effects of zinc in micropropagated plants of Paulownia tomentosa

    Ben Bahri et al.http://www.openaccessscience.com

    [email protected]

    membrane is particularly sensitive to heavy

    metal toxicity since membrane permeability and

    thus, its functionality can be affected by altering

    the intrinsic membrane protein such that H+

    -

    ATPases (Hall 2002). This stress results in a

    production of reactive oxygen species (ROS).

    Overproduction of these ROS leads to oxidativedamage in plant tissues due to lipid peroxidation

    leading to the formation of degradative products

    such as alkanes and aldehydes (Ferrat et al.

    2003). Therefore, toxic symptoms such as

    chlorosis, growth retardation, browning of roots

    and a cell cycle arrest can be observed. To fight

    this oxidative stress, plants produce defense sys-

    tems enzymatic and non enzymatic antioxidants

    in order to regulate the concentration of ROS

    and thus maintain ion homeostasis within cells

    (Clemens 2001, Hall 2002).

    Zn levels in vitro plants

    The Zn content in the vitro plantlets was

    measured at the end of the experiment. As

    shown in Fig.3, the Zn levels recorded in vitro

    plantlets vary from 121 mg g-1

    in the control to

    2580 mg/g in vitro plants grown in the medium

    containing the highest concentration of Zn

    (1000 M).

    0

    500

    1000

    1500

    2000

    2500

    3000

    Znlevels(g/gD

    0 200 400 600 800 1000

    Zn concentration (M)

    Figure 3. Zn levels (g g-1

    DW) on tissues ofvitro plantlets Paulownia tomentosa (Thunb) Steud.

    Indeed, Zn was mostly stored in the tissues

    plantlets ofPaulownia, where the accumulation

    was almost linearly related with the Zn concen-

    tration in the MS medium. Similar results were

    reported by Azzarello et al. (2012) whose de-

    tected a linear increase of Zn accumulation in

    Paulownia tomentosa plants with the 1000 M

    of Zn. At highest zinc concentrations (2000,

    3000 and 5000 M), a linear decrease of Zn ac-

    cumulation was detected and was clearly linked

    to the reduced biomass production.

    On the other hand, Marques et al. (2006)

    showed that Solanum nigrum accumulated up to

    1450, 3240 and 3810 mg Zn kg-1

    in the leaves,

    stems and roots, respectively but without any

    visual toxicity signs. This confirmed the idea

    that Paulownia tomentosa is Zn tolerant and

    hyperaccumulator plant.

    Conclusion

    In order to evaluate the tolerance and the

    ability to Zn accumulation in Paulownia

    tomentosa grown under in vitro conditions, dif-

    ferent morphological parameters as well as

    analysis of the Zn content in tissues of plantlets

    were determined. Results showed that 1000 M

    Zn concentration caused a reduction of about18.86% and 50.5%, respectively on shoot height

    and dry biomass compared to the control. This

    decline was more pronounced in the root sys-

    tem. However, a significant reduction in the

    number of roots was noticed and estimated by

    84.9% with 1000 M Zn concentration com-

    pared to the control. The high levels of Zn in the

    culture medium limited the root formation.

    However, the determination of Zn levels

    showed good accumulation in the tissues of

    plantlets with ranging from 121 g g-1

    DW to2580 g g-1 DW. Overall, results showed that

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    Int. J. Med. Arom. Plants Effects of zinc in micropropagated plants of Paulownia tomentosa

    Ben Bahri et al.http://www.openaccessscience.com

    [email protected]

    Paulownia tomentosa could be considered as a

    bio-indicator and Zn accumulator medicinal

    plant since it can tolerate and accumulate high

    Zn content.

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