Banana is an important group of plants that provides millions of livelihood.  India has a rich genetic diversity of banana with more than 90 distinct clones. Banana is grown under diverse conditions and production systems and hence selection of varieties is based on needs and situation. Around 20 cultivars viz. Dwarf Cavendish, Robusta, Monthan, Poovan, Nendran, Red banana, `Safed Velchi, Basrai, Ardhapuri, Rasthali, Karpurvalli, Karthali, Grande Naine, Malbhog etc. are commercially cultivated. Malbhog is a Musa cultivar of AAB genomic group under subgroup of silk type having reference accession number TRY0077 (Daniells et al., 2001) commonly found in Assam, India. Malbhog banana is very popular and has high commercial value in Assam due to its sweet aroma, taste and high post-harvest life. However, shortage of quality planting materials remains the bottleneck for the banana farmers of the state.  Tissue culture techniques have been employed for large scale banana planting material production. Many regeneration protocols dealing with different Musa species has been reported for in vitro propagation of banana and plantains (El-Saghir,1997; Kodym and Zapata-Arias, 2001; Vidhya and Nair, 2002; Madhulatha et al., 2004; Sebastian and Mathew, 2004; Anilkumar and Sajeevan, 2005; El-Dougdoug et al., 2006; Kulkarni et al., 2006; Darvari et al., 2010; North et al., 2012; Ngomuo et al., 2014; Qamar et al., 2015). However establishment of aseptic culture remain a great challenge for banana micropropagation. Endo-bacterial contamination is one of the major problems for aseptic establishment of banana where the contaminants may survive in the plant material for several subculture cycles and it may over extend the period of time without expressing symptoms in the tissue or visible signs in the medium (Van Den Houwe and Swennen, 2000). The use of antibiotic, commercial fungicide, thermotherapy, warm water treatment and ultrasonic treatment are some of the commonly used methods to control in vitro contamination (Cole, 1996). In this report, osmotic stress induction of banana suckers as a technique to reduce microbial contamination and its subsequent influence on multiplication is emphasized.

Stress induction

Two months old sword suckers of banana variety Malbhog were collected from farmer’s field of Assam, India. Sucker were washed thoroughly under running tap water for half an hour and stress was induced by keeping the cleaned suckers in shade and fresh weight were recorded at 0, 7, 14, 21, 28 days interval to ascertain loss of moisture. Loss of moisture was calculated as follows:

Moisture loss (%): a-b/a x 100

where, a = initial fresh weight of suckers; b = fresh weight of suckers after stress for d.

Establishment of explants in aseptic media

After each interval of stress induction, 25 suckers were washed under running tap water for 30 min and trimmed into square block ranging from 5 to 8 cm in sizes and dipped in fungicide solution (2 gm/l Bavistin and 100 mg/l ascorbic acid) for 30 min followed by washing with liquid detergent (Extran, Merck, 0.05 ml/l) for 20 min and 4 to 5 times rinsed with clean water. Thereafter, sucker cubes were treated with Savlon (Johnsons and Johnsons) for 30 min and brought under laminar hood. Surface sterilization was carried out with 0.1 mg/l HgCl₂ fortified with 2 to 3 drops of Tween 20 (Merck) for 10 to 12 min and the disinfectant was removed by 4 to 6 rinse with sterile distilled water. After surface sterilization the cut ends of explants were trimmed with a sterile surgical blade and the shoot tips of 8 to 10 mm from decapitated shoot apexes of suckers are carefully inoculated in pre sterilized MS medium (Murashige and Skoog, 1962) supplemented with 1 to 2 mg/l 6-Benzyl-aminopurine (BAP), 2-4 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l α-naphthalene acetic acid (NAA), 30 g  /l  sucrose, 2.2 g /l Gelrite. The pH of the medium was adjusted to 5.8 using 0.1 M NaOH before autoclaving for 20 min at 121°C at 15 lbs psi pressure. The inoculated media with explants were incubated in the growth room at 25±2°C with 16 h illuminations. The influence of stress on contamination reduction was calculated as follows:

Contamination % = c-d/c*100

where c = contamination of explant of fresh suckers d = contamination of sucker explants stressed for d.

Plant multiplication

Aseptically established explants were subcultured into basal medium (MS) with varying combination of 0.5 to 1 mg/l of BAP, 0.5 mg/l  NAA and 2 to 4 mg/l  2, 4-D. Number of plantlets from each explant was recorded for subsequent subculture till 6^th passage. Subculture was carried out at every 3 weeks interval.

Root induction and hardening

Microshoots of 4 to 5 cm length were separated from the cluster and inoculated into MS medium supplemented with 0.5 mg/l IAA. Rooted plantlets of 6 to 8 cm length were taken out from culture bottle and washed under running tap water for 10 min to remove media attached to root system. Thereafter plantlets were treated with 1 g/l Bavistin for 5 min and transferred to polybag (size 12 × 18 cm) containing river sand and kept inside the polyhouse for 21 days. Subsequently, these plantlets were transferred to larger polybag (size 15 × 21 cm) containing soil, seasoned cow dung at 1:1 and kept under agro shade net house with provision of 50% penetration of natural light. Irrigation was ensured depending on the requirements.

Banana is cultivated in humus and organic matter rich soil where microbial load in rhizosphere region is very high and poses challenge to establish aseptic culture. Various techniques to contain contamination begin with pretreatment of donor plants to reduce contamination (Holdgate and Zandvoort, 1997) with antibiotics and fungicides (Kritzinger et al., 1997) as well as anti-microbial formulations, such as plant protection mixture (PPM) (Guri and Patel, 1998). The explants are also culture indexed for contamination by standard microbio-logical techniques, which are occasionally supplemented with tests based on molecular biology or other techniques (George, 1993; Leifert and Woodward, 1998; Leifert and Cassells, 2001). Since plants do not have an immune system to antibiotics and as such many of the antibiotics, that are effective against bacteria, fungi, and phy-toplasmas, are toxic to plants as well.  Use  of  antibiotics is not full proof or the desired method to get rid of microbial contamination (Pierik, 1989). Islam and Zobayed (2000) reported microbial contamination in banana using sugar free medium. We report inexpensive and effective mode of microbial contamination reduction due to stress induction in suckers. Besides reducing contamination, this technique also has the advantage of stretching inoculation period after material collection.

Plants cell experience severe strain and stresses during multiplication of plantlets under artificial medium depending on the hormonal combination. Thus, synergistic effect of BAP and auxin on shoot proliferation has already been reported in banana tissue culture (El-Saghir, 1997; Nauyen and Kozai, 2001; Kagera et al., 2004; Kalimuthu et al., 2007; Sheidai et al., 2008; Qamar et al., 2015). In the present experiment, plants tissue were exposed to osmotic stress during pre-inoculation stage and its subsequent exposure to hormonal combina-tion in the artificial medium responded exceptionally well for microbial contamination reduction and multiplication of microshoots. This is the first report to study the influence of stress on contamination free in vitro establishment and multiplication of microshoots (Tables 1 and 2). Root induction is one of the important steps in plant tissue culture. In case of banana, rooting occurs simultaneously for 4 weeks old cultures. However, micro shoots when subcultured in MS medium fortified with 0.5 mg/l IAA induced 9 numbers of healthy roots per shoot within 9 days of culture. Rooting of microshoot in IBA supple-mented media has been reported (Haq and Dahot, 2007).

Hardening of plantlets in two stages was found beneficial. Initially, plantlets were transplanted into river sand with intermittent watering for 21 days in polyhouse followed by subsequent transplanting into larger polybag with river sand and seasoned cow dung in agroshade resulted to satisfactory survival and growth. 

Microbial contamination in tissue culture could be curtailed for sucker based banana explant by inducing stress for 21 days storage in shade. This technique also increases the multiplication efficiency of microshoots in the subsequent subculture. The present protocol with 21 days osmotic stress induction of banana suckers with MS medium fortified with BAP (1.0 mg/l) and NAA (0.5 mg/l) for in vitro establishment, shoot initiation, mass scale multiplication, rooting and two step hardening could be used for mass scale production of banana planting material. 

The author(s) did not declare any conflict of interest.

Authors are thankful to Dr. R. K. Pachauri, Director General, The Energy and Resources Institute for his encouragement and support. Authors are also thankful to Department of Science and Technology, Government of India, for the research grant vide DST No: SR/SO/PS-47/06 dated 10-9-2007.