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A study on callus induction of Ipomoea mauritiana: an Ayurvedic medicinal plant.

INTRODUCTION

Ipomoea mauritiana (commonly termed as 'Giant Potato' in English and 'BhuiKumra' in Bengali) is a woody vine in the family Convolvulaceae, which has reports on wide range of therapeutic uses in the Indian sub-content. The tuber of the plant is rich in carbohydrate, phytosterol, glycosides, flavonoids and saponins [13]. 'Vidari is an Ayurvedic raw drug where tuberous root of the plant and root parts are used to increase appetite, as a galactagogue, in anti-aging medicine, and as a stimulant, carminative, and tonic [37]. Roots of the plant are used for blackening the hair and as well as to treat sexually transmitted diseases for men in Bangladesh [30]. Reportedly, it hasantioxidant-properties [7]; roots are used to increase strength for both men and women [31]. Paste of the tubers also has been used in the treatment of skin diseases, anorexia, fevers, and burning sensations, and used to increase breast milk production [18]. Moreover, its tubers have analgesic activity [19] and hepatoprotective activity [33].

Since the last few decades, the techniques of plant tissue culture have developed as a new and powerful tool for crop improvement [6]. Among the techniques, micropropagation plays a unique role in conservation of species, particularly those having medicinal value [2]. Technology for culture of plant cell, tissue and organ is important for the production of necessary metabolites [43]. Exogenous application of phythormones can generate callus induction which may help to increase availability of plant material for in vitro studies. Somaclonal variations, somatic embryogenesis, organogenesis and isolation of different economically valuable phytochemicals can be obtained from callus culture [11,29,17,25,4].

Biotechnological approaches like tissue culture, callus culture, cell suspension culture, organ culture, elicitor treatment and tissue engineering are important methods for industrial production of bioactive compounds [39,22,32]. Among them, in vitro production of secondary metabolites plays a vital role for the manufacture of therapeutic drugs, nutraceuticals, flavoring agents, agrochemicals, colors, biopesticides, and food additives in large quantities by the pharmaceutical industries and herbal companies [5]. For example, different studies for isolation of secondary metabolites has been conducted on Artemisia annua for artemisinin [3], Aspidospermaramiflorum for ramiflorin [26], Camellia chinensisfor flavones [23], Capsicum annum for capsaicin [40,38], and Centellaasiatica for asiaticoside [16]. The aims of the present study wereto establish an efficient protocol for callus culture with different concentrations of phytohormones on I. mauritiana and observed growth of callus and biomass in vitro.

Materials and Methods

Explant source and preparation:

One year aged plants of Ipomoea mauritiana were selected for this experiment which was grown in the Garden of Medicinal Plants at the Department of Biotechnology and Genetic Engineering, University of Development Alternative, Dhaka. The donors were taxonomically identified from Bangladesh National Herbarium, Dhaka. Nodal explants were used and dissected from the juvenile stems (2-3 cm) of the donors for callus initiation. The explants were washed under running tap water for 40 minutes and then treated with Tween-80 for 7 minutes as surfactant. The explants were ready to culture in Murashige and Skoog (MS) medium [21].

The culture medium compositions for induction of callus were prepared as MS medium containing different concentrations (see detail in Results section) of phytohormones including 6-benzylaminupurine (BAP); kinetin (Kn); 2,4-dichlorophenoxy acetic acid (2, 4-D); [??]-naphthaleneacetic acid (NAA); indole-3-acetic acid (IAA), indole-3-butyric acid (IBA); sucrose (3%) and agar (7%) after surface sterilization of stems with 0.1% Hg[Cl.sub.2]. 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/[cm.sup.2].

Culture of explants:

Cultures were maintained at 26 [+ or -] 2[degrees]C with a photoperiod of 12/12 h under an illumination of 20 imol [m.sup.-2][s.sup.-1] provided by cool white fluorescence lamps.

Data recording and analysis:

The percentage of explants (%) creating acalli, its structure, color, texture, width (a) and length (b) in mm was determined for 4 weeks and tabulated for detailed study. The callus diameter (d) was then calculated as per d = [square root of ((a x b))], as described by Farshadfar et al. [10].

Results:

Callus initiation:

Nodal explants responded in callus induction medium when MS was supplemented withBAP[1.5 and 2.0 mg/l] within 20days of inoculation. Callus was green, granular and hard in texture on MS media supplemented with 1.5 mg/l BAP (Table 1).Profuse callogenesis was observed when MS medium was enriched with 2, 4-D. It showed a good callus diameter and as well as loose calluses on medium (Graph 7). The calluses were morphologically granular in MS + 2, 4-D [1.0 mg/l] (Figure G).

Effect of BAP:

2.0 mm size callus was harvested from initial calluses generated in [MS + 1.5 mg/l BAP] and then sub-cultured in to the different concentrations (2, 3, 4, and 5 mg/l) of BAP. Greenish white and granular callus was shown in 2 mg/l BAP whereas granular but light brown callus was found in 3mg/l BAP. We found that increased concentration of BAP can result in changes of callus color. BAP [4.0 mg/l and 5.0mg/l] showed whitish brown and friable, and whitish green (60%), brown (40%) and friable calluses, respectively (Figure A). The textures remained hard in the MS medium supplemented with BAP. Mean diameter of callus in BAP showed a trend of fluctuation with changes of media composition. Callus diameter found in 2.0 mg/l BAP was significantly increased with 3.0 mg/l and 5.0 mg/l but not with 4.0mg/l BAP (Graph 1).

Graphical Representations:

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Effect of auxin and cytokinin in combination:

When callogenic responses were studied in MS +2, 4- D [0.5 mg/l], the 2.0 mm sized calluses were separated and then sub-cultured in MS media with three combinations of BAP and 2, 4-D [1.0 mg/l BAP + 0.5 mg/l 2, 4- D; 1.5 mg/l BAP + 0.5 mg/l 2, 4- D; 2 mg/l BAP+ 1.0 mg/l 2, 4- D]. All the combinations showed similar responses in color (brown), morphology (granular) and texture (loose) after 28 days of inoculation (Table 1, Figure 2).Callus growth rates were changed with elevation of both 2, 4-D and BAP, measured as the mean callus diameter in mm (Graph 2). Highest callus mean diameter was obtained in 2, 4-D [0.5 mg/l] + BAP [1.0 mg/l] and had significant differences with callus mean diameterswithother combinations.

The change of BAP concentration (1.0 to 2.5 mg/l) in combination with constant NAA [0.5 mg/l] in MS medium showed differences in callus colors and textures (Table 1). The morphology of calluses was granular and embryonic (100%) in every medium composition. The colors of the calluses were changed from greenish brown to brown with the increase of BAP along with NAA [0.5 mg/l] (Figure C). Similarly, when BAP level was increased, it showed that textures were medium hard to fragile and then compact. Changes of callus diameter was only raised when BAP was increased from 1.0 mg/l to 1.5 mg/l with constant NAA [0.5 mg/l] but remained steady when BAP was supplemented with >2.0 mg/l in MS medium (Graph 3).

Greenish brown callus was obtained from a combination of BAP and IAA in MS medium (Figure D). The textures of the callus were hard (100%) and granular in these medium compositions (Table 1). Callus diameter significantly increased when both BAP [2.0 mg/l] and IAA [0.8 mg/l] was increased (Graph 4).

Kn and NAA showed prolific callus growth where the changes of Kn resulted in changes in callus morphology from non-embryonic to embryonic (Figure E). Increase of Kn also showed changes in callus texture. The calluses were greenish brown (50%) and dark brown (50%) in Kn [1.0 mg/l] + NAA [0.5 mg/l] and turned to greenish when Kn was increased (Table 1). Surprisingly, the growth of callus and its diameter significantly increased at highest concentration of Kn [2.0 mg/l] in MS (Graph 5).

Effect of IRA on root induction from callus:

IBA was effective in granular and embryonic callus induction in the present study. The callus diameter reduced with increases in IBA, but there were no significant differences among the treatments (Graph 6). The most distinguishable observation was these compositions could induce rhizogenesis in calluses (Figure F). The color of the calluses varied from greenish brown to brown and whitish green at low, medium and highest concentration of IBA fortified MS medium (Table 1).

[ILLUSTRATION OMITTED]

Discussion:

In Vitexnegundo, highest callogenic effect was also found when MS medium was supplemented with 2, 4-D [2.0 mg/l] [8], which is similar to our findings.Combinations of BAP and 2,4-D were found effective in callus growth of Abrusprecatorius by Hasan et al. [12] and Sen et al. [34] also reported fastest callus growth in Achyranthesaspera, which is similar to our current study. The combination of BAP and NAA hada positive effect on callus induction of this plant;a similar effect was observed by Yasmin et al. [42] in potato plant. Noor et al. [24] observed BAP + IAA to be effective in callus induction and as well as development of green embryonic structures in wheat cultivars which is in harmony with the current findings. Callus growth was improved in Kn + NAA in the present study and also found effective in Matthiolaincana from leaf micro-cuttings by Kaviani et al. [14]. Root induction from calluses was reported in Gerbera jamesonii [27], which was similar to our current experiment with IBA enriched in MS medium.

Conclusion:

The current study revealed that callus culture of I. mauritiana depended on the role of phytohormones and composition of culture medium. The callus growth can be varied on the concentration of a specific hormone and it also showed varied morphology, texture and color. The developed method could a play a significant role on in vitro callus induction of this species and isolation of potential phytochemicals by different pharmaceutical and herbal industries.

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Saiful Islam, MastaHasenur Reza Chawdhury, Imam Hossain, Md. SaydurRahmanSayeed, ShahnazRahman, F. M. SafiulAzam and Mohammed Rahmatullah

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

Received: 25 April 2014; Revised:: 20 May 2014; Accepted: 25 May 2014; Available online: 28 June 2014

Corresponding Author: Professor Dr. Mohammed Rahmatullah, Pro-Vice Chancellor, University of Development Alternative, House No. 78, Road No. 11A (new), Dhanmondi R/A, Dhaka-1205, Bangladesh.

Ph: 88-02-9136285 Fax: 88-02-8157339 E-mail: rahamatm@hotmail.com
Table 1: Effect of Phyotohormones in callus induction and
their characteristics.

   MS + Hormonal concentration (mg/l)

BAP     Kn    2, 4-D   NAA   IAA   IBA

1.5
2.0
3.0
4.0
5.0
1.0            0.5
1.5            0.5
2.0            1.0
1.0                    0.5
1.5                    0.5
2.                     0.5
2.5                    0.5
1.5                          0.2
1.5                          0.8
2.0                          0.8
        1.0            0.5
        1.5            0.5
        2.0            0.5
                                   0.5
                                   1.0
                                   1.5
               0.5
               1.0

        Color *        Morphology           Texture

BAP

1.5     GR, 100%     Granular, 100%       Hard, 100%
2.0     GR, 11%      Granular, 100%       Hard, 100%
        WG, 89%
3.0     LB, 100%     Granular, 100%       Hard, 100%
4.0     WB, 100%     Friable, 100%         Hard 100%
5.0     WG, 60%      Friable, 100%        Hard, 100%
        BR, 40%
1.0     BR, 100%     Granular, 100%       Loose, 100%
1.5     BR, 100%     Granular, 100%       Loose, 100%
2.0     BR, 100%     Granular, 100%       Loose, 100%
1.0     GB, 100%      Granular and      Medium Compact,
                    Embryonic, 100%          100%
1.5     BR, 20%         Granular         Fragile, 100%
        LB, 80%     Embryonic, 100%
2.      LB, 100%        Granular         Fragile, 100%
                    Embryonic, 100%
2.5     BR, 40 %        Granular        Hard, 50% Medium
        GB, 60%     Embryonic, 100%      Compact, 50%
1.5     GR, 100%     Granular, 100%       Hard, 100%
1.5     GB, 100%     Granular, 100%       Hard, 100%
2.0     GB, 100%     Granular, 100%       Hard, 100%
        GB, 50%      Embryonic, 50%        Hard, 50%
        DB, 50%    Non-Embryonic, 50%     Loose, 50%
        GR, 100%    Embryonic, 100%       Hard, 100%
        GB, 100%    Embryonic, 100%       Hard, 100%
        GB, 100%    Embryonic, 100%       Rooted, 30%
        BR, 100%     Granular, 100%
        WG, 100%    Embryonic, 100%       Rooted, 20%
        WB, 100%     Granular, 100%       Loose, 100%
        WB, 40%      Granular, 40%        Loose, 100%
        LB, 60%    Non-embryonic, 60%

* GR = Greenish, WG = Whitish Green, LB= Light Brown,
WB = Whitish Brown, BR=Brown, GB = Greenish Brown,
DB = Dark Brown.
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Title Annotation:Research Article
Author:Islam, Saiful; Chawdhury, Masta Hasenur Reza; Hossain, Imam; Sayeed, Saydur Rahman; Rahman, Shahnaz;
Publication:American-Eurasian Journal of Sustainable Agriculture
Article Type:Report
Geographic Code:9BANG
Date:Apr 1, 2014
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