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

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


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, 1987Leifert 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 ster-ilants 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 Acid and a solution of Ascorbic acid and citric acid 100 mg/L ( Table 1) for 10 minutes after sterilization and trimming further in sterile environment. After that explants were taken out from the solution and washed with sterile water and then treated with Sodium Hypochlorite solution (0.5-1.0 %) for different period of exposure (7, 10, and 15 minutes) followed by 4 times washing with sterile water ( Table 2) . The treated suckers were further pealed up by removing one more scale and treated with 0.1 % HgCl 2 for different period of exposure (5 and 7 minutes) and washed with sterile water for 4 times. Finally the final trimming was done to a size of 2.0 cm and dipped in a sterile solution of FL Cystine (15 mg/L) for 30 minutes and then explants were directly inoculated in four different MS media viz., Bi1: MS Basal, Bi2: MS+BAP 0.2 mg L -1 , Bi3: MS +BAP 0.3 mg L -1 and Bi4: MS +BAP 0.5 mg without washing and incubated in the Plant Growth Room (PGR) at 25 0 C ± 2 0 C with 16 hours illuminations and 8 hours dark phases. Contamination percentage at weekly interval and microshoots production at 2 weeks intervals were recorded and the contaminated cultures were discarded immediately and autoclaved at 121 0 C and 15 lbs p.s.i pressure for 1 hour.

RESULTS
The present study was conducted to standardize the best sterilization protocol for in vitro propagation of Musa cv. Amritsagar, Malbhog and Chenichampa. Different pre-treatment methods were tested to prevent browning of explants before surface sterilization and after sterilization. Treatment combination Ascorbic acid (100 mg/l) + Citric acid (100 mg/l) for 1 hour before surface sterilization and 10 minutes after sterilization (PT4) gave the best results with regard to number of days for initial browning of explants and days required for first subculture (Table 3). For sterilization of explants two different chemicals i.e. sodium hypochlorite (0.5 % and 1.0 %) and Mercuric chloride (0.1%) were used for the present study with treatment duration of 7, 10, and 15 minutes for sodium hypochlorite and 5 and 7 minutes for Mercuric chloride respectively.
In this study, the treatment combination T10, T9, T6 and T8 for both Amritsagar and Malbhog were found to be the best combination with regard to achieving highest percentage of contamination free healthy culture, whereas in case of Chenichampa treatment combination T10, T9, T6 and T5 gave the best result following the disinfection procedure described in the materials and methods section. T10 gave the best results with regard to per cent of health culture establishment with 85, 75, and 90 per cent for Amritsagar, Malbhog and Chenichampa respectively (Table 5). Axillary buds showed cent percent viability along with the emergence of 2-3 shoots per node within four weeks of initiation (Plate 3).
Plate 3: Axillary buds with emergence of 2-3 shoots per node within four weeks of initiation

ReseaRch PaPeR
Effect on infection of cultures: Result showed that with increase in exposure time the infection was decreases in both the chemicals. The infection was notably much lower in the NaOCl (1.0 %) and HgCl 2 (0.1%) with 15 and 7 minutes duration (T10) respectively. The single treatment either with NaOCl or HgCl 2 showed higher infection (Table 5, T1  T2  T3  T4  T5  T6  T7  T8  T9  T10  T1  T2  T3  T4  T5  T6  T7  T8  T9  T10  T1  T2  T3  T4  T5  T6  T7  T8  T9  T10 Amrit Sagar Malbhog Chenichampa Cultuvar wise treatment Contamination % Figure 1: Effect of sterilants on contamination per cent Effect on healthy cultures (overall survivals): The data depicted in the table 5 indicate that with the increase in concentration and exposure duration of both the chemicals the survival rate was also increased (Figure 2). The survival obtained with 15 minute of NaOCl followed by 7 minutes of HgCl 2 (T10) treatment was significantly higher than all other concentration and exposure duration of both the chemicals.

DISCUSSION:
The use of field grown plants as a direct source of explants for the production of 'clean' in vitro plantlets, presents a major challenge with regard to microbial contaminations during the process of initiation and maintenance of viable in vitro cultures. 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, 1987Leifert et al., 1990), the majority of which is caused by fungal, yeast and bacterial contaminants (Leifert, et al., 1994).
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 and Sharma 1988;Habiba et al., 2002;Molla et al., 2004, Titov et al., 2006. Double disinfection method has also been applied by some researchers, where first large size explants are disinfected, followed by shoot tip excision and finally disinfection by some other chemical agents (Silva et al., 1998;Nandwani et al., 2000;Rahman et al., 2002;Madhulatha et al., 2004). Sometimes explants are treated with fungicides and antibiotics to minimize the contamination load in in vitro cultures (Van den Houwe 1998;Nandwani et al., 2000). Ethanol has also been used by a number of research workers for disinfection purposes (Silva et al., 1998;Rahman et al., 2002;Jalil et al., 2003).
Onuoha et al., (2011) achieved the contamination free Plantain culture (100%) in the explants treated with HgCL 2 for 6 min. Houwe et al., (1998) reported that treatment of shoot tips with Rifampicin at 100 mg. l -1 during 1 month resulted in 100 % bacteria free explants without any phytotoxity. Amongst the two sterilants i.e. NaOCl and HgCl 2 , NaOCl was found better for controlling the infection and it had not any adverse effect on explant even in long duration (15 minutes). Sodium hypochlorite at higher concentration (1.0 %) has turned out to be a better sterilant than mercuric chlorite alone at 0.1 % for 5 minutes treatment time. However, a treatment combination of Sodium hypochlorite (1.0%) for 15 minutes followed and HgCl 2 (0.1%) for 7 minutes resulted the highest percentage (85, 75 and 90 %) of aseptic culture establishment in banana cultivars Amritsagar, Malbhog and Chenichampa respectively in in vitro condition followed by Sodium hypochlorite (1.0%) for 10 minutes and HgCl 2 (0.1 %) for 7 minutes and Sodium hypochlorite (1.0%) for 15 minutes alone.