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Volume 6, Issue 5, October Issue - 2018, Pages:890-894


Authors: Ningthoujam Sandhyarani Devi, RK Imotomba, Rocky Thokchom
Abstract: For the micropropagation of Curcuma angustifolia usually rhizome buds are used as explants which are definitely associated with various types of soil-born microorganisms, therefore only one type of sterilant cannot be used for the sterilization of these rhizome buds. Therefore present investigation was carried out to develop the most effective surface sterilization protocol for C. angustifolia. In the present study, explants were treated with three sterilizing agents viz., fungicide (Dhanustin) at 1 % (m/v), 70 % (v/v) ethanol and mercuric chloride (HgCl2) at a concentration of 0.1 % (m/v). Among the different used sterilants with varying treatment time, 1 % (m/v) fungicide with 4 drops of Tween 20 for 30 minutes followed by 70 % alcohol for 1 minute and lastly 0.1 % (m/v) HgCl2 for 10 minutes was found most effective surface sterilizing combination for C. angustifolia micorpopagation.
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Full Text: 1 Introduction Curcuma angustifolia Roxb. belongs to the family Zingiberaceae and it distributed throughout central, southern and eastern India but most commonly reported from the Northeast and Western Coastal Plains of India (Srivastava et al., 2006; Sharma, 2012). It is generally known as East Indian Arrowroot due to its heritage being considered from the Indian subcontinent. Further, it is also known by various names in different languages such as ‘Narrow-leaved turmeric’ in English, ‘Tikhur’ in Hindi and ‘Yaipan’ in Manipuri. The rhizome contributes a major role in its nutritional and medicinal field. This rhizomatous plant is used to ease cough and heal bronchitis and is highly effective on diarrhoea and dysentery (Patel et al., 2015). The essential oil extracted from various plant parts also has antifungal and antibacterial activities (Doble et al., 2011). Further, Shukla et al. (2011) reported antimicrobial properties of C. angustifolia leaf essential oil. Phytochemical constituents of C. angustifolia are studied by various researchers and reported the presence of ar-curcumin, β-pipene, α-terpineol, camphor, zingirol and borneol (Banerjee et al., 1980). However, Nguyen et al. (2001) reported more than 30 compounds from dried and fresh rhizome of C. angustifolia. Jena et al. (2017) also reported the presence of curzerenone, cadinene and γ-endesmol acetate as the main constituents in leaf oil and curzerenone, camphor and germacrone as major component in rhizome oil. Besides having various medicinal values, biochemical constituents  are less exploited. In Manipur, the flowers of this herb are also used as a delicacy in various dishes due to its distinct intriguing taste and it is popularly used in a chutney known as ‘Eromba’. This medicinal herb is naturally propagated mainly through rhizomes and in vitro propagation techniques via plant tissue culture. Natural propagation method of Curcuma species are very slow process compared to the opportunity offered by plant tissue culture techniques. Tissue culture protocol for various Curcuma species such as C. amada (Borthakur & Bordoloi, 1992; Nayak, 2001; Prakash et al., 2004; Shukla et al., 2007), C. aromatica (Nayak, 2000), C. longa (Nadgauda et al., 1978; Balachandran et al., 1990, Salvi et al., 2002; Rahman et al., 2004;Prathanturarug et al., 2005), C. zedoaria (Loc et al., 2005) and C. spp. (Yasuda et al., 1988; Tyagi et al., 2004) has been developed by various researchers. For a successful and effective micropropagation method, an efficient explant sterilization step should be established (Traore et al., 2005). Surface sterilization of the naturally contaminated living plant materials from the environment is a critical step since it involves the use of chemical solutions like sodium hypochlorite, ethanol, mercuric chloride etc. that are toxic to the plant tissues (George, 1993). Types, amount and exposure time of the sterilants are important for the establishment of a contamination free sterilization protocol. Higher concentration of these sterilants is also responsible for lower growth and viability of explants. Therefore, present investigation has been conducted to develop a simple surface sterilization protocol for in vitro culturing of C. angustifolia. 2 Materials and Methods 2.1 Plant Materials Plants of C. angustifolia (Figure 1A) were collected from Bishnupur District of Manipur (23o50’-24o43’N;92o58’-94o45’E) and maintained in the Botanical Field of Pandit Deen Dayal Upadhyay Institute of Agricultural Sciences, Utlou, Manipur. The plants were identified taxonomically by experts. 2.2 Culture Medium For micropropagation, MS medium (Murashige & Skoog, 1962) containing 3 % (w/v) sucrose and 0.8 % (w/v) Agar (Hi-media, Mumbai, India) fortified with various concentration and combination of α-nephthalene acetic acid (NAA), 6-benzylamino purine (BAP) were used. All the growth regulators were purchased from Sigma (St. Louis, MO, USA). Prior to gelling with agar the pH of the medium was adjusted to 5.8 using 1 N NaOH and 1 N HCl and sterilized by autoclaving at 121oC and 1.05 Kgcm-2 pressures for 20 mins. 2.3 Explant sterilization The rhizome axillary buds were cut into small blocks having a bud each. Before surface sterilization the explants were washed in Tween 20 detergent solution for 30 min and further rinsed with tap water followed by treatment with 1(m/v) % Dhanustin (fungicide) for 30 min and again rinsed with distilled water for 3-4 times.The rhizome buds were trimmed into smaller sized cubes under the Laminar Air Flow chamber and subjected to different treatment duration regime of 70 % (v/v) alcohol and 0.1 % HgCl2(m/v). In the first experiment the buds were treated with 70 % (v/v) alcohol for 0.5 minutes followed by rinsing 2-3 times with sterilized distilled water and finally treated with 0.1 % (m/v) HgCl2 subsequently for 5,7 and 10 minutes. Later on exposure time for 70 % alcohol was increased to 1 minute followed by 0.1 % (m/v) HgCl2 for 5,7, 10 and 12 minutes. 3 Results and discussion In this experiment, C. angustifolia was subjected to eight different treatment regimes using MS (Murashige & Skoog, 1962) medium supplemented with different concentrations and combinations of naphthalene acetic acid (NAA) and benzyl aminopurine (BAP). Effect of exposure time duration and various combinations of sterilants on explants contamination (%) and survival                        rate of the explants (%) have been represented in Table 1. Highest   A
C
D
B
    percentage of contamination (100%) was observed when the explants were treated with 0.1 % (m/v) HgCl2 for 5 minutes followed by 70 % (v/v) alcohol for 30 seconds. At this concentration,  cultures got bacterial (Figure 1B) as well as some fungal contamination (Figure 1C) which may be because of less treatment time with HgCl2 and 70 % alcohol. The highest survival percentage (100%) was achieved when explants were subjected to Tween 20 and 1 % (m/v) fungicide solution for 30 minutes followed by treatment with 70 % (v/v) alcohol for 1 minute then washing with 0.1 % (m/v) HgCl2 for 10 minutes which showed fresh and vigorous growth (Figure 1D). It was observed that treatment with 70 % alcohol less than 1 minute showed higher contamination compared to treatment for 1 minute. This may be because of weaker chemical effect due to shorter exposure duration that was unable to kill the pathogens present in the explant. Highest mortality percentage was obtained when the explants were treated with 0.1 % HgCl2 for 10 minutes. Treatment with 0.1 % HgCl2 for more than 10 minutes resulted into death and desicated cultures. The use of mercuric chloride as a sterilizing agent has been frequently reported (Naika & Krishna, 2008; Preethi et al., 2011; Anburaj et al., 2011). However, exposure of HgCl2 had negative effects on survival rate of explants (Danso et al., 2011). Longer period of exposure with HgCl2 leads the browning of explants and death. Our report had the same parallel effect with the previous studies (Wesely et al., 2011; Johnson et al., 2011). Surface sterilization of the plant materials from the environment is a critical step since it involves the use of chemicals like fungicide, ethanol and mercuric chloride which are toxic to the plant’s tissues. Plant species and type of explants are important for the development of proper sterilization protocol. The surface contaminant varies based on the type of natural environment they are inhabiting and age of the plant materials. For in vitro cultures to avoid contamination it is an essential step to standardize an effective sterilization protocol for proper in vitro culturing. Conclusion Sterilization of the rhizome axillary buds of C.angustifolia with 70% (v/v) alcohol and 0.1% (m/v) HgCl2 is very critical as evidenced from this study. Our results show that a combination of 70 % (v/v) alcohol for 1 minute and 0.1 % (m/v) HgCl2 for 10 minutes is the most effective surface sterilization protocol for in vitro micropropagation of C. angustifolia. As very limited reports on in vitro micropropagation, chemical profile and molecular studies are available for this medicinally important plant, there is a great requirement for further investigation. Conflicts of interest No conflicts of interests are declared by authors for the contents in this manuscript.
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