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Impact of Various Factors on in Vitro Investigation of Rare Mangrove Species Sonneratia Apetala Buch.-Ham

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Impact of Various Factors on in Vitro Investigation of Rare Mangrove Species Sonneratia Apetala Buch.-Ham Brazilian Journal of Biological Sciences, 2016, v. 3, no. 5, p. 181-191. ISSN 2358-2731 http://dx.doi.org/10.21472/bjbs.030516 Impact of various factors on in vitro investigation of rare mangrove species Sonneratia apetala Buch.-Ham. (Myrtales: Lythraceae) and Suaeda maritima (L.) Dumort (Caryophyllales: Amaranthaceae) Abdul Kader¹, Sankar Narayan Sinha² and Parthadeb Ghosh¹,* ¹Cytogenetics & Plant Biotechnology Research Unit. Department of Botany. University of Kalyani. Kalyani - 741235, Nadia. West Bengal, India. *Email: [email protected]. ²Environmental Microbiology Research Laboratory. Department of Botany. University of Kalyani, Kalyani -741235, Nadia. West Bengal, India. Abstract. Recent studies have showed the importance and destruction of mangroves. So their restoration through tissue culture Received study is urgently required because in vivo propagation is plagued Jan. 1, 2016 with unforeseen obstacles. In vitro investigation of mangroves found to be suitable material for salt tolerant mechanism studies and anti Accepted stress gene isolation. This study describes for the first time in vitro May 30, 2016 approach for rare species Sonneratia apetala Buch.-Ham. (Myrtales: Released Lythraceae) and herb species Suaeda maritima (L.) Dumort June 30, 2016 (Caryophyllales: Amaranthaceae) through callus. For this investigation, as a source of explants various part (leaf, intermodal Open Acess Full Text Article and nodal segments) of both mangroves were used. These explants were cultured in various types of media (MS, LS, WPM, X and B5) with different combination of phytohormones (2, 4-D and NAA with BAP in combination). We also examined the effect of NaCl and seasons on callus initiation and growth. The highest rate of callus formation was obtained with nodal explants in MS medium supplemented with 1.5 mg.L-¹ NAA and 0.5 mg.L-¹ BAP in combination and 1 mg.L-¹ NAA and 0.5 mg.L-¹ BAP in combination for Sonneratia apetala and Suaeda maritime, respectively. We also found that callus initiation rate and growth decreased with increasing NaCl concentration higher than 80 mM and 120 mM for Sonneratia apetala and Suaeda maritima, respectively in MS media. This study also found that monsoon season was best time for in vitro investigation of mangroves. The results presented here give an insight into the development of in vitro investigation suitable for mangroves. The initiated callus could be restored in low salaine or devoid of salaine land. Keywords: Culture media screening; Callus culture; Mangrove; Salt tolerance; Sonneretia apetala; Suaeda maritima. Introduction subtropical muddy beaches worldwide. It is considered as low-cost and high-efficiency Mangrove is a typical ecosystem in wetland systems for the treatment of the intertidal area from tropical to municipal wastewater (Tam and Wong, ISSN 2358-2731/BJBS-2016-0001/3/5/16/181 Braz. J. Biol. Sci. http://revista.rebibio.net 182 Kader et al. 1996) and for remediation of heavy metal- species of mangrove (Tian et al., 2009, polluted marine environments (MacFarlane Naskar and Bakshi, 1987, Ren et al., 2009). et al., 2003). It has critical ecosystem This species is reported to improve soil functions such as coastal protection, land fertility and having medicinal property like stabilization and CO2 fixation (Ren et al., antioxidant, antimicrobial and cytotoxic 2009). However destruction of mangrove activity (Ren et al., 2009). The leaf of this forest is noticed due to natural and species is used to treat hepatitis (Banerjee et anthropogenic uses (Alongi, 2002). For al., 2008, Jaimini et al., 2011). The fruit of mangrove restoration many have planted this species is consumed by local people of propagules and seeds which depend on mangrove region after cooking or other natural yields. Therefore it is not possible to preparations. Additionally ripe fruits are get enough numbers of good propagules used to treat parasites and half ripe fruit for and seeds all over the year. Recently there cough (Banerjee et al., 2008 and Jaimini et is shortage of mangrove seed source al., 2011). Shete et al. (2007) reported that (Komiyama et al., 1996). In these the aerial part of this species has the circumstances, the alternative method is potentiality for the bioaccumulation of Pb tissue culture study to get enough number and Zn. of micropropagated plantlet. Mangrove The Suaeda maritima (L.) Dumort species are physiologically unique in their (Caryophyllales: Amaranthaceae) is an adaptations to such water logged and saline annual, succulent, salt-marsh mangrove condition. Crop scientists, studying the annual herb, with semi-cylindrical leaves unique adaptation pattern of mangroves, are from Chenopodiaceae family grows in very keen to impart these unique characters in alkaline and saline moist soils (Polić et al., food crops by breeding or biotechnological 2009, Ravikumar et al., 2011). It is found in means (Fukomoto et al., 2004) as salinity the sea cost of Asia, Europe, North and and water logging are among the major South America and Australia too (Polić et environmental threats with serious al., 2009). The leaf of this species is used to implication on food, fuel and fibre treat hepatitis and having medicinal production, especially in arid and semiarid properties like antiviral, antibacterial, regions (Dagar, 2005). Besides, about one- antioxidant and hepatoprotective activity third of all agricultural lands are becoming (Singh et al., 2012). saline (Dagar, 2005). To understand the salt Keeping the deforestation, tissue and water logging tolerance theoretically or culture problem, medicinal and economical biochemically, callus or cell culture of value of these plants in mind, we described mangroves may provide promising result. a preliminary study of micropropagation However there are scanty literatures are and degree of salt tolerant, through callus available on some mangrove species culture, for preservation and production of because they are much recalcitrant for micropropagated plant for future restoration tissue culture study (Fukomoto et al., 2004, at degraded areas. To our knowledge, this is Kawana and Sasamoto, 2008). During in the first report of in vitro investigation of vitro studies explants frequently turn brown these species with systematic study. or black shortly after inoculation and result in tissue death (Kathiresan, 1990) due to Materials and methods presence of high amount of tannin and phenolic compounds in mangroves (Ravi Plant material and Kathiresan, 1990). Different explants from mature The Sonneratia apetala Buch.- plants were collected at various seasons all Ham. (Myrtales: Lythraceae) is a true over the year from Gosaba region mangrove tree from Sonneratiaceae family (88° 39’ 46” E and 22° 15’ 45” N) of Indian found in all over the mangrove region of the Sundarban Mangrove Forest. world and are considered as invasive Braz. J. Biol. Sci., 2016, v. 3, no. 5, p. 181-191. Impact of various factors on in vitro investigation of rare mangrove species 183 weight. For this study we observed two Surface sterilization of explants auxin like 2, 4-dichloro phenoxy acetic acid The explants (1-1.5 cm long) were (2,4-D) and α-naphthalene acetic acid washed with 2 % (v/v) teepol for 10 min (NAA) in combination with one cytokinins and rinsed thrice with distilled water, i.e, 6-benzyladenine purine (BAP). disinfected with 0.15 % (w/v) HgCl2 for 10 min and again rinsed four times with Data collection and statistical sterile distilled water. There after the analysis sterilized explants were cut into small Each experiment was repeated three segments and aseptically inoculated on times with 13 replicates. Data were different nutrient media. All the analyzed by one way analysis of variance experimental procedures were carried out (ANOVA) and the difference between under aseptic conditions in a laminar means were scored using Duncan’s airflow. Multiple Range Test P ≤ 0.05 (Duncan, 1955) on the statistical package of SPSS Culture conditions (Version 10). The cultures were generally maintained at standard condition with a Results temperature of 25 ± 1 °C under 65 ± 5% relative humidity and 16 h photoperiod Selection of explants for callus under 2000 lux intensity provided by white initiation fluorescent lamps. Among the different explants used, leaves were not found to be suitable for Culture medium and growth callusing. Callus was obtained from nodal regulators used and internodal segments (Figure 1 and 2). Four different types of media were used for callus initiation like Murashiege Screening for suitable culture and Skoog (MS) medium (Murashiege and media and hormonal combinations skoog, 1962), Woody Plant medium Callus initiation was observed (WPM, Lloyd and McCown, 1981), within 2 to 3 weeks after inoculation using Linsmaier and Skoog (LS) medium different combinations of auxin (NAA) and (Linsmaier and Skoog, 1965) and X cytokinin (BAP) in all the media however medium (Rao et al., 1998). Considering the X medium required 4 to 5 weeks for callus herb species Suaeda maritima in mind we initiation for both species (Figure 1 and 2). also included B5 (Gamborg et al., 1968) The highest rate of callus formation was medium along with other mentioned obtained in MS medium supplemented with medium. All media were supplemented 1.5 mg L-1 NAA and 0.5 mg L-1 BAP in with 3% (w/v) sucrose and 0.8% agar combination and 1 mg L-1 NAA and powder. The pH of the medium was 0.5 mg L-1 BAP in combination for adjusted to 5.7 before autoclaving. For S. apetala (Table 1) and S. maritima (Table induction of callus and determining the 2), respectively. We also tried to initiate degree of salt tolerant mechanism, NaCl callus on 2, 4-D alone or in combination was added in the medium at various with BAP but there were no response for S. concentration of this experiment. The callus apetela. The 2,4-D containing medium initiation rate (the ratio of the number of showed low callus initiation rate, slow explants pieces having calli to total number growth and dark brown nature callus for of explants pieces planted in the same S.
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