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In Vitro Sterilization Protocol for Micropropagation of Musa (AAA group) ‘Amritsagar’ Musa (AAB group) ‘Malbhog’ and Musa (AAB group) ‘Chenichampa’ Banana

N. Goswami,P. Handique

Published 2011 in Indian journal of applied research

ABSTRACT

Sterilization procedure was standardized for three popular banana cultivars viz., Musa (AAA group) ‘Amritsagar’; Musa (AAB group) ‘Malbhog’; and Musa (AAB group) ‘Chenichampa’. Two sterilants, sodium hypochlorite (0.5 % and 1.0 %) and mercuric chloride (0.1 %) were used both singly and in combination for surface sterilization of explants under different exposure time ranging from 5 to 15 minutes. Sterilized explants were inoculated on four different MS media viz., MS Basal, MS+BAP 0.2 mg L-1, MS +BAP 0.3 mg L-1 and MS +BAP 0.5 mg L-1 to evaluate the response of different chemicals. The observations were recorded regularly up to 30 days with respect to the dead cultures, infected cultures and healthy cultures. Result showed that a treatment combination of Sodium hypochlorite (1.0%) for 15 minutes followed by HgCl2 (0.1%) for 7 minutes gives the highest percentage of aseptic culture establishment in in vitro condition. INTRODUCTION In vitro propagation technique for banana involves various steps i.e. selection of explants (suckers), its sterilization, initiation and establishment, shoot proliferation and rooting of microshoots. The first condition for the success of in vitro propagation is the getting aseptic culture. The maintenance of aseptic or sterile conditions is essential for successful tissue culture procedures. To maintain an aseptic environment, all culture vessels, media and instruments used in handling tissues, as well as explant itself must be sterilized. The importance is to keep the air, surface and floor free of dust and it is require carrying out all the operation in laminar airflow sterile cabinet. The use of field grown plants as a direct source of explant material for the production of ‘clean’ in vitro plantlets, presents a major challenge. Microbial contaminations are the major hurdle to the initiation and maintenance of viable in vitro cultures. Explant contamination occurs due to several plant and environmental related factors such as plant species, age of the plant, explant source and prevailing weather condition. Despite the best timing and selection efforts it is difficult to eliminate contamination from in vitro grown plants. Losses due to contamination in in vitro condition average between 3 and 15% at every subculture in the majority of commercial and scientific plant tissue culture laboratories (Boxus & Terzi, 1987, 1988 ; Leifert et al., 1990), the majority of which is caused by fungal, yeast and bacterial contaminants (Leifert, et al., 1994). For in vitro culture initiation, explants are normally collected from field grown plants, so the plant material is liable to be contaminated by microorganisms which must be disinfected before explants are transferred to in vitro conditions. Variations in sterilization procedure have been proposed by several researchers. Sodium hypochlorite is the most commonly used disinfectant for surface sterilization of banana explants (Cronauer and Krikorian 1984; Mendes et al., 1996; Muhammad et at., 2004). Some other investigators have replaced sodium hypochlorite with low concentration of mercuric chloride (Banerjee & Sharma 1988; Habiba et al., 2002; Molla et al., 2004, Titov et al., 2006). Sterilization is the process of making explants contamination free before in vitro establishment of cultures. Various sterilization agents are used to decontaminate the tissues. These sterilants are also toxic to the plant tissues, hence proper concentration of sterilants, duration of exposing the explant to the various sterilants, the sequences of using these sterilants have to be standardized to minimize the injury to the explant for achieving better survival rate. Two different chemicals i.e. sodium hypochlorite (0.5 % and 1.0 %) and Mercuric chloride (0.1%) were used for the study to standardize the best sterilization protocol for in vitro propagation of Musa cv. Amritsagar (AAA), Malbhog (AAB) and Chenichampa (AAB). MATERIAL AND METHOD The present study was carried out at Department of Biotechnology, Gauhati University, Assam and at The Energy and Resources Institute, Guwahati, Assam with the objective to evaluate the effect of different sterilants at different concentrations and different exposure time on banana explants for in vitro propagation. Three banana cultivars viz., Amritsagar (AAA), Malbhog (AAB) and Chenichampa (AAB) were considered for the research programme. These cultivars have economic importance to the people of the state. Amritsagar (AAA) is good table banana cultivars and fairly resembles the internationally reputed banana Gros Michel, which once occupied 63% of the world market. Plants are medium sized, fruit size good, rind medium thick and the ripe banana develops a bright yellow colour. Malbhog (AAB) is one of the most popular table banana cultivar indigenous to Assam and has a high demand on market due to its sweet aroma, taste and higher post harvest life. It is a medium tall cultivar flowers in 18 months. Cheni Champa (AAB) is one of the hardiest medium tall banana cultivar. The plant is resistant to Fusarium wilt and fairly resistant to bunchy top disease. Fruits are small in size with thin peel, creamy pulp and sub-acid taste. The pre treated suckers of all the three cultivars were washed under running tap water for 30 minutes to 1 hour to remove the dirt (Plate 1). Thereafter suckers were trimmed into 3-4 inched sizes (Plate 2). The trimmed explants were further treated with Savlon (Johnsons & Johnsons) for 15 minutes. Thereafter, explants containing meristem and rhizomatous base were treated with a mixture of 2 % Sodium Hypochlorite + Captan or Dithane M-45 1 g/L of water and Rifampicin (0.1%) for 45 minutes (Plate 3). Tween-20 was added as wetting agent to enhance maximum penetration of sterilizing agents. After that explants were rinsed with clean water for 4 times and a quick dip (15 sec) in 70 % alcohol was given before transferring the explants in sterile environment (Laminar Air Flow Cabinet). Explants were then dipped in double distilled water, Ascorbic Acid, Citric Acid and a solution of Ascorbic acid and citric acid 100 mg/L for 1 hour before surface sterilization and 50 mg/L Ascorbic Acid, 50 mg/L Citric

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