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Studies with callus induction of Vitex negundo: an aromatic medicinal plant.


Vitex negundo Linn. (syn: V. inesia Lam.), locally termed as Nishinda (in Bengali) is a deciduous, woody aromatic and multipurpose medicinal shrub belonging to the Verbenaceae family. It is usually found in South-Asian countries. (Watt, 1972; Nasir and Ali, 1974; Wealth of India, 1976). The major form of healthcare in the rural parts of developing countries is traditional medicine, which is mostly plant-based (Jager and van Staden 2000). V. negundo have diverse medicinal uses in the folk medicinal system of Bangladesh (Rahmatullah et al., 2010; Khan and Rashid, 2006).

Along with the utilization in traditional medicine by local practitioners and healers, this plant also reportedly showed diverse pharmacological properties including analgesic (Zheng et al., 2009); antinociceptive (Zheng et al., 2010); antiinflammatory (Yunos et al., 2005; Jana et al., 1999; Dharmasiri et al., 2003, Tandon, 2006a); anti-fertility (Das et al., 2004); anti-feedant (Chandramu et al., 2003; Sahayaraj, 1998); anti-histamine (Dharmasiri et al., 2003; Tiwari and Tripathi, 2007); antioxidant (Vishal and Gupta, 2005; Rooban et al., 2009); antihyperglycemic effect (Villasenor and Lamadrid, 2006); cytotoxicity for human cancer cell line (Diaz et al., 2003); hepatoprotective activity against liver damage induced by d-galactosamine (Yang et al., 1987), commonly used tubercular drugs (Tandon et al., 2008) and carbon tetrachloride (Tasduq et al., 2008, Raj et al., 2008); laxative activity (Adnaik et al., 2008); immunomodulatory effect (Ravishankar and Shukla, 2007); and mosquito repellant effect (Karunamoorthi et al., 2008; Hebbalkar et al., 1992).

The plant parts are reported to have anti-microfilarial (Sahare et al., 2008a; Sahare et al., 2008b); anti-viral (Woradulayapinij et al., 2005; Nguyen-Pouplin et al., 2007); anti-bacterial (Samy et al., 1998); anti-fungal (Guleria and Kumar, 2006; Sathiamoorthy et al., 2007; Aswar et al., 2009); insecticidal (Paneru and Shivakoti, 2001; Raja et al., 2000; Rajendran, and Sriranjini, 2008; En-shun, 2009); larvicidal (Nathan et al., 2006; Kamaraj et al., 2008; Karmegam et al., 1997; Kannathasan et al., 2007; Kannathasan et al., 2008; Rahuman et al., 2009; Pushpalatha and Muthukrishnan, 1995; Yuan et al., 2006); as well as significant effect on antagonizing the Vipera russellii and Naja kaouthia venom induced lethal activity in both in vitro and in vivo studies (Alam and Gomes, 2003). The plant is reported to contain potent and novel therapeutic agents for scavenging of NO and the regulation of pathological conditions caused by excessive generation of NO and its oxidation product, peroxynitrite (Jagetia and Baliga, 2004). Administration of V. negundo extracts also potentiated the effect of commonly used antiinflammatory drugs (Tandon et al., 2006b); analgesics (Gupta et al., 2005); sedative-hypnotic drugs (Gupta et al., 1997; Gupta et al., 1999); and anti-convulsive agents (Tandon and Gupta, 2005). Inhibitory effect of V. negundo against active enzymes has also been observed for lipoxygenase and butyryl-cholinesterase (Azhar-Ul-Haq et al., 2004); a-chymotrypsin (Lodhi et al., 2008); xanthine-oxidase (Umamaheswari et al., 2007a), and tyrosinase (Azhar-Ul-Haq et al., 2006).

Callus is a de-differentiated state of tissue through the exogenous application of plant growth hormones in vitro. This callus can solve the problem of unavailable plant materials for in vitro studies. Different physiological and morphogenic responses can also be observed through callus culture like somaclonal variations, somatic embryogenesis, organogenesis; it can also pave the way for isolating economically valuable phytochemicals, which can avoid the collection of plant materials from natural sources (Flick et al., 1983; Raghavan, 1986; Larkin and Scowcraft, 1981; Ogita et al., 2009; Berkov et al., 2009). In the areas of plant biotechnology, callus and cell culture carries a special role for producing medicinal and bioactive compounds in large-scale from plants (Taha et al., 2008; Rao and Ravishankar, 2002). Phytochemicals are serving as a major source of pharmaceuticals, flavors, agrochemicals, colors, biopesticides, and food additives (Bourgaud et al., 2001). In this perspective, callus culture already has been studied for different medicinal plants like Artemisia annua for artemisinin (Baldi and Dixit, 2008), Aspidosperma ramiflorum for ramiflorin (Olivira et al., 2001), Camellia chinensis for flavones (Nikolaeva et al., 2009), Capsicum annum for capsaicin (Varindra et al., 2000; Umamaheswari and Lalitha, 2007b), and Centella asiatica for asiaticoside (Kiong et al., 2005). We intended to develop a suitable protocol for studying the callus culture of V. negundo through this research. Our objective was to investigate the effects of phytohormones for callus induction and to observe how the plant materials perform after the exogenous application of plant growth regulators in the culture medium on this species.

Materials and methods

Explant source and preparation:

Donor V. negundo plants (4 years old), grown in the Garden of Medicinal Plant at the Department of Biotechnology and Genetic Engineering, University of Development Alternative were used in the study. Plant was taxonomically identified by Bangladesh National Herbarium. For callus initiation, young leaves (YL) and internodal segments (IN) of young vegetative stem with a size of 1.5-2 cm were excised from the ex vitro plant and washed under running tap water for 30 minutes and then with Tween-80. All the explants were cultured on basal Murashige-Skoog (MS) medium (Murashige, T. and F. Skoog, 1962) containing different concentrations (see detail in Results section) of phytohormones including 6-benzylaminupurine (BAP); 2,4 dichlorophenoxy acetic acid (2, 4-D), a-naphthalene acetic acid (NAA); sucrose (3%) and agar (7%) after surface sterilization with 0.1% HgCl2 and rinsing four times in sterile distilled water. The pH of all media was adjusted to 5.8 before autoclaving and media was sterilized by autoclaving for 20 min at 121[degrees]C and 1.05 kg/cm2.

Culture of explants

Cultures were maintained at 26 [+ or -] 2[degrees]C with a photoperiod of 16/8 h under an illumination of 20 imol m-2s- 1 provided by cool white fluorescence lamps.

Data recording and analysis

The percentage of explants (%) creating a calli, its structure, color and texture was determined for 4 weeks and tabulated for detailed study.

Results and discussion

Effect of 2,4-D:

Within two weeks of culture period, 100% callogenic response was achieved when the medium was fortified with 2,4-D [2.0 mg/l] for both types of explants, leaves and internodes (Table 1), (Figure 1). Other treatments with this synthetic hormone showed at least 60% responses. All the hormonal concentrations in this experiment formed whitish friable calluses. Each responded explant with a concentration range of [2.0 mg/l] to [3.0 mg/l] of 2,4-D developed calli faster than others and the callus was vigorous in growth. Results from this experiment revealed that both types of explants responded equally in every medium composition.


Effect of NAA:

Good callogenic response was found in MS + NAA [1.5 mg/l] (Figure 2). All treatments with NAA resulted in formation of friable and embryonic callus with greenish white color in the medium composition as described in Table 2. Quick callus growth was obtained with concentrations of NAA at [2.0 mg/l] and [2.5 mg/l]. No further morphogenic response was observed when the cultures were maintained up to eight weeks in the medium.

Synergistic Effect of 2,4-D and NAA:

White colored callus appeared when the explants were cultured in MS medium supplemented with combinations of 2,4-D and NAA. Good growth of calluses were seen when the concentrations of both the auxins were high. Fastest callogenic response was observed when 2,4-D [3.0 mg/l] and NAA [1.0 mg/l] was added to MS medium (Table 3), (Figure 3). The noticeable characteristic of the responded callus was the variation of texture in callus along with changes of hormonal combinations. It varied from compact to loose, and then to loose with wet surfaces. Leaf and nodal explants did not show any appreciable differences.



Synergistic effect of 2, 4-D and BAP:

Only higher combinations of both 2,4-D and BAP formed better calli but turned brownish within three weeks of culture (Figure 4). The texture was loose with wet surfaces. Callus growth was not prominently increased or showed variations along with the change of concentrations. The best medium found from these synergistic experiments was 2, 4-D [1.0mg/l] and BAP [2.0 mg/l] fortified MS medium (Table 4).



Though 2,4-D is a synthetic auxin, its role in callus induction was particularly observed in our experiment. Efficacy of exogenous 2, 4-D has also been reported with other medicinal plants. Results described by Rani et al. (2003), Thomas and Maseena (2006), Hassan et al. (2009) were also in agreement with our result for using this synthetic plant growth regulator in the culture medium for Withania somnifera, Cardiospermum halicacabum Linn and Abrus precatorious, respectively. We have noted that the effect of 2,4-D was explant independent for V. negundo, whereas Hassan et al. (2009) found the best response only from young leaves with same textures of calluses. Use of single auxin for induction of calli was also confirmed in our experiment when the medium was enriched with NAA alone in MS. Such finding has also been reported by Nurazah et al. (2009) when MS medium was fortified with NAA [3.0 mg/l] within 4 weeks of Cananga odorata culture. Both Nurazah et al. (2009) with C. odorata, and Sirchl et al. (2008) with Garcinia mangostana observed no callus formation in absence of NAA. Synergism of 2,4-D and NAA found by the authors also agree with results obtained by Nikolaeve et al. (2009) with Camellia chinensis. However, effectiveness of auxin-cytokinin combination for callus induction found in this present study differ from the findings of Olivira et al. (2001), Kiong et al. (2005), and Baldi and Dixit (2008).

In conclusion, the following points may be mentioned--the effectiveness of callus induction depends on the type of growth regulators used and explants source; the characteristics of the callus (color and texture) depend on culture medium; and the protocol of callus induction of V. negundo opens new vistas that could facilitate phytochemical production and extraction of pharmaceuticals from the callus without harvesting the plant itself.


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Farzana Begum Chowdhury, F.M. Safiul Azam, Md. Maruf Hassan, Farhana Israt Jahan, Anita Rani Chowdhury, Syeda Seraj, Zubaida Khatun, Mohammed Rahmatullah

Faculty of Life Sciences, University of Development Alternative, Dhanmondi, Dhaka-1205, Bangladesh.

Corresponding Author: Mohammed Rahmatullah, University of Development Alternative Dhanmondi R/A, Dhaka1205, Bangladesh.

Pon: 88-02-9136285; Fax: 88-02-8157339

Table 1: Effect of 2,4/D (mg/l) in MS medium for callus induction of
V. negundo.

MS + Hormone    Source of    Number of             Percentage
(mg/l) 2, 4-D   explants     explants inoculated   of response (%)

0.5             Leaf         5                     60
                Internodes   5                     60
1.0             Leaf         5                     80
                Internodes   5                     80
1.5             Leaf         5                     60
                Internodes   5                     60
2.0             Leaf         5                     100
                Internodes   5                     100
2.5             Leaf         5                     60
                Internodes   5                     60
3.0             Leaf         5                     80
                Internodes   5                     80

MS + Hormone    Source of    Callogenesis   Time required
(mg/l) 2, 4-D   explants                    for response (day)

0.5             Leaf         0              14
                Internodes   0              14
1.0             Leaf         0              12
                Internodes   0              12
1.5             Leaf         0              8
                Internodes   0              8
2.0             Leaf         +++            8
                Internodes   +++            8
2.5             Leaf         0              11
                Internodes   0              11
3.0             Leaf         0              11
                Internodes   +++            11

MS + Hormone    Source of    Color   Texture
(mg/l) 2, 4-D   explants

0.5             Leaf         White   Friable
                Internodes   White   Friable
1.0             Leaf         White   Friable
                Internodes   White   Friable
1.5             Leaf         White   Friable
                Internodes   White   Friable
2.0             Leaf         White   Friable
                Internodes   White   Friable
2.5             Leaf         White   Friable
                Internodes   White   Friable
3.0             Leaf         White   Friable
                Internodes   White   Friable

-No response, + Poor response, ++ Good response, +++ Better response

Table 2: Effect of NAA (mg/l) in MS medium for callus induction of V.

MS+ Hormone   Source of   Number of             Percentage of
(mg/L)        explants    explants inoculated   response (%)

0.5           Leaf        5                     00
              Internode   5                     60
1.0           Leaf        5                     60
              Internode   5                     100
1.5           Leaf        5                     100
              Internode   5                     100
2.0           Leaf        5                     60
              Internode   5                     60
2.5           Leaf        5                     100
              Internode   5                     100
3.0           Leaf        5                     60
              Internode   5                     80

MS+ Hormone   Source of   Callogenesis   Time required
(mg/L)        explants                   for response (day)

0.5           Leaf        -              28
              Internode   0              12
1.0           Leaf        0              20
              Internode   0              18
1.5           Leaf        0              14
              Internode   +++            11
2.0           Leaf        0              10
              Internode   0              10
2.5           Leaf        0              10
              Internode   0              10
3.0           Leaf        0              10
              Internode   0              8

MS+ Hormone   Source of   Color            TextureNAA
(mg/L)        explants

0.5           Leaf        ----             ----
              Internode   Green            Friable
1.0           Leaf        Greenish white   Friable
              Internode   Green            Friable
1.5           Leaf        Greenish white   Friable
              Internode   Greenish white   Friable, embryonic
2.0           Leaf        Green            Friable
              Internode   Green            Friable
2.5           Leaf        Green            Friable
              Internode   Greenish white   Friable
3.0           Leaf        Greenish white   Friable
              Internode   Greenish white   Friable

-No response, + Poor response, ++ Good response, +++ Better response.

Table 3: Effect of 2,4/D + NAA (mg/l) in MS medium for callus
induction of V. negundo.

MS+ Hormone     Source of   Number of    Percentage
(mg/l) 2, 4-D   explants    explants     of response (%)
+ NAA                       inoculated

0.5+1.0         Leaf        5            80
                Internode   5            80
0.5+1.5         leaf        5            60
                Internode   5            80
1.0+1.0         leaf        5            80
                Internode   5            100
1.0+1.5         Leaf        5            80
                Internode   5            100
1.5+1.0         leaf        5            60
                Internode   5            60
1.5+1.5         Leaf        5            100
                Internode   5            80
2.0+1.0         Leaf        5            60
                Internode   5            60
2.0+1.5         Leaf        5            60
                Internode   5            80
2.5+0.5         Leaf        5            100
                Internode   5            100
2.5+1.0         Leaf        5            100
                Internode   5            100
2.5+1.5         Leaf        5            80
                Internode   5            60

2.5+2.0         Leaf        5            60
                Internode   5            60
3.0+0.5         Leaf        5            60
                Internode   5            80
3.0+1.0         Leaf        5            60
                Internode   5            60
3.0+1.5         Leaf        5            60
                Internode   5            80
3.0+2.0         Leaf        5            80
                Internode   5            80

MS+ Hormone     Source of   Callogenesis    Time required
(mg/l) 2, 4-D   explants                    for response (day)

0.5+1.0         Leaf        +++             9
                Internode   +++             9
0.5+1.5         leaf        0               10
                Internode   +++             10
1.0+1.0         leaf        +++             10
                Internode   +++             10
1.0+1.5         Leaf        +++             10
                Internode   +++             9
1.5+1.0         leaf        0               9
                Internode   +++             9
1.5+1.5         Leaf        0               9
                Internode   0               9
2.0+1.0         Leaf        0               7
                Internode   0               8
2.0+1.5         Leaf        +++             7
                Internode   0               8
2.5+0.5         Leaf        0               8
                Internode   +++             7
2.5+1.0         Leaf        0               8
                Internode   +++             8
2.5+1.5         Leaf        0               9
                Internode   +++             9

2.5+2.0         Leaf        0               13
                Internode   0               13
3.0+0.5         Leaf        0               8
                Internode   +++             8
3.0+1.0         Leaf        +++             7
                Internode   +++             7
3.0+1.5         Leaf        0               8
                Internode   +++             8
3.0+2.0         Leaf        0               10
                Internode   +++             10

MS+ Hormone     Source of   Color            Texture
(mg/l) 2, 4-D   explants

0.5+1.0         Leaf        White            Loose
                Internode   White            Compact
0.5+1.5         leaf        White            Loose
                Internode   White            Compact
1.0+1.0         leaf        White            Loose
                Internode   White            Loose
1.0+1.5         Leaf        White            Loose and wet
                Internode   White            Loose and wet
1.5+1.0         leaf        White            Loose and wet
                Internode   White            Loose and wet
1.5+1.5         Leaf        White            Loose and wet
                Internode   White            Loose and wet
2.0+1.0         Leaf        White            Loose and wet
                Internode   White            Loose
2.0+1.5         Leaf        White            Loose
                Internode   White            Loose
2.5+0.5         Leaf        White            Loose
                Internode   Yellowish        Loose
2.5+1.0         Leaf        White            Loose
                Internode   White            Loose and wet
2.5+1.5         Leaf        White            Loose
                Internode   Greenish white   Loose and wet
                                             Loose and wet
2.5+2.0         Leaf        White            Loose
                Internode   White            Loose
3.0+0.5         Leaf        White            Loose
                Internode   White            Loose and wet
3.0+1.0         Leaf        White            Loose and wet
                Internode   White            Loose and wet
3.0+1.5         Leaf        White            Loose and wet
                Internode   White            Loose and wet
3.0+2.0         Leaf        White            Loose and wet
                Internode   White            Loose and wet

-No response, + Poor response, ++ Good response, +++ Better response.

Table 4: Effect of 2,4/D (mg/l) and BAP combination in MS medium for
callus induction of V. negundo.

MS + Hormone        Source of    Number of           Percentage
(mg/l) 2,4-D+ BAP   explants     explants included   of response (%)

1.0+0.5             Leaf         5                   60
                    Internodes   5                   80
1.0+1.0             Leaf         5                   80
                    Internodes   5                   80
1.0+1.5             Leaf         5                   80
                    Internodes   5                   100
1.0+2.0             Leaf         5                   100
                    internodes   5                   100

MS + Hormone        Source of    Callogenesis   Time required
(mg/l) 2,4-D+ BAP   explants                    for response (day)

1.0+0.5             Leaf         0              12
                    Internodes   0              10
1.0+1.0             Leaf         0              12
                    Internodes   0              7
1.0+1.5             Leaf         0              10
                    Internodes   0              11
1.0+2.0             Leaf         0              11
                    internodes   0              11

MS + Hormone        Source of    Color     Texture
(mg/l) 2,4-D+ BAP   explants

1.0+0.5             Leaf         Yellow    Loose and wetly
                    Internodes   Yellow    Loose and wetly
1.0+1.0             Leaf         Brown     Loose and wetly
                    Internodes   Brown     Loose and wetly
1.0+1.5             Leaf         Brown     Loose and wetly
                    Internodes   Brown     Loose and wetly
1.0+2.0             Leaf         Yellow    Loose and wetly
                    internodes   Yellow    Loose and wetly

+ Poor response, ++ Good response
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Title Annotation:Original Articles
Author:Chowdhury, Farzana Begum; Azam, F.M. Safiul; Hassan, Maruf; Jahan, Farhana Israt; Chowdhury, Anita R
Publication:American-Eurasian Journal of Sustainable Agriculture
Article Type:Report
Geographic Code:9BANG
Date:Jan 1, 2011
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