Chitosan as a Growth Promoter and Enhance Survival Rate in an in vitro Culture of Banana (Musa spp.) Cultivar ‘Bantala’

Banana (Musa spp.) is the herbaceous monocotyledonous plant and it belongs to the Musaceae family. It’s mostly cultivation in tropical and subtropical regions worldwide and in India, Kerala, Karnataka, Gujarat, Orissa, Bihar, Eastern Uttar Pradesh, West Bengal, Assam, North Eastern states are the major producers (Saravanapandeeswari and Vanitha 2018). The banana developed more than one hundred twenty nations covering just about ten million hectares with 95 million tons generation of one year. On the basis of production of food crops, the rice is at 1^st, the wheat at 2^nd, the maize at 3^rd rank (Rout et al., 2000) and followed by banana remains at the 4^th rank (Saravanapandeeswari and Vanitha 2018). Initially, banana generation started from Southeast Asia and after that production of this species gradually spread whole over India (Chaurasia et al., 2017). Above ‘970’ indigenous banana were collected from India and kept in the National Research Centre on Banana (NRCB), Tiruchirappalli, Tamil Nadu, India. China acquires the top position in fruit producer but the second position in India around the world but in the case of banana, India top most banana production than China. The total annual production of fruits has been estimated to be 88.97 metric tons (MT) from an area of 6.38 Million ha (Rathod and Mishra, 2018). To demonstrated and found the no somaclonal variation as well as free from viral contamination of the explants from both sucker and rhizome of banana explants (Kacar et al., 2012). The sucker was used for the propagation of banana plantlets through vegetative methods but its slow production, time-consuming as well as cause diseases and very poor preservation (Hussein, 2012). The microrhizome based clonal propagation is already reported in Curcuma caesia Roxb (Sarma et al., 2021).The production of good quality banana crops affects viral diseases through non-professional cultivation (Wambugu et al., 2008). An in vitro culture was better than a conventional method for the propagation of banana plants because of genetic uniformity, a short period, high plantlet production, disease-free and round the year (Ortiz and Vuylsteke, 1996). The propagation of banana plants through tissue culture techniques 30% higher than the conventional method (Pradeep et al., 1992). A single plant or a small piece of tissue (explants) can be produced a large number of uniform disease-free plants through in vitro culture method (Borah et al., 2019). Chitosans are linear polysaccharides. It’s made from chitin shells of shrimp, crabs and other crustaceans with an alkaline substance and is also found in both fungal and insect cell walls (Walker-Simmons et al., 1983). It’s also the antifungal agent against plant disease resistance and non-toxic effect on the environment (Vander et al., 1998; Benhamou et al., 1994; El-Ghaouth et al., 1994, Cassells et al., 1999; Roby et al., 1987 and Tiuterev et al., 1996). Chitosan has so many properties like growth stimulator, antimicrobial properties, plant disease resistance, biodegradability, biocompatible for the environment, etc (Uthairatanakij et al., 2007). This study is based on the effect of various phytohormones and chitosan with MS medium for the propagation of bantala variety of banana plants by in vitro culture and prevents fungal and bacterial infections.

Explants
 
The investigation was carried out from 2019-2020, in the department of plant tissue culture (PTC), KIIT School of Biotechnology, Bhubaneswar and Odisha.
       
Samples were collected from the Orissa University of Agriculture and Technology (OUAT), Bhubaneswar, Odisha. The rhizome and sucker as explants of banana cv. Bantala were selected. The rhizome and sucker were washed thoroughly under running tap water for two hours. The plant materials were dipped into 1% laboline for fifteen to twenty min. The explants were cleaned thoroughly with sterilized H₂O and exposed to UV in laminar airflow for fifteen minutes. The explants were dipped in 70% ethyl alcohol for 60seconds. Then immediately transferred into sterile glass materials and pour 0.1% mercuric chloride (HgCl₂) for ten to fifteen minutes with frequent shaking. The materials were washed thoroughly with sterile distilled water three to four times. Then finally, the explants were dried on sterile filter papers and cut into small in cube size of 0.5 cm and ready for inoculation on MS medium.
 
Culture condition
 
The plant’s growth and development in various stages such as the response of plants, shoot initiation, shoot multiplication and rooting used the full-strength and half-strength MS (Murashige and Skoog, 1962) media (HiMedia, PT101) with plant growth regulators (PGRs) and Chitosan. CleriGarTM (Himedia, PCT0902) was added at a range of 0.35%-0.5% (w/v) as gelling agents. MS medium was added by various concentrations of plant growth regulators. The prepared media has adjusted the pH 5.8 by 0.1N NaOH or HCL and sterilized in autoclaves at 121°C and 15 psi for twenty minutes. Then the prepared explants were inoculation in MS medium. All cultures were carried out into the culture room. The cultures were incubated at 25±2°C and 16/8 hrs photoperiod using cool-white fluorescent lights (50 µmoL m^-2s^-1) and the culture room maintained 60% of relative humidity.
 
Chitosan solution preparation
 
The preparation of a stock solution (100 mg/L) of chitosan powder (Himedia, TC242) was dissolved by 4 M HCL. The adjustment of pH value before the slurry was added into the media.
 
Phytohormones and Chitosan trial concentrations range
 
BAP: 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 mg/L
IAA: 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 mg/L
IBA:  0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 mg/L
Chitosan: 0.0, 5.0, 10, 15, 20, 25, 30, 35, 40 and 45 mg/L
 
The response of rhizome and sucker explants
 
There were used different combination concentrations of BAP, IAA, chitosan and full-strength MS medium in rhizome and sucker explants. Both explants were tried 100 combination concentrations separately for the response of explants. To observed the data based on the visual gradation i.e. inoculated explants initially swelling and after greenish color as considered but black in color plants discarded. The data scored after one week from inoculation.
 
The response, shoot initiation, shoot proliferation and rooting
 
The selected best 10 numbers of concentrations were used for the experiments. To follow the response, shoot initiation and proliferation medium (full-strength MS with BAP, IAA and chitosan) after completed the response stage of explants in both rhizome and sucker. To removed the unwanted shoot parts from the cut of initiated explants and transferred the explants to a multiplication medium (full-strength MS with BAP, IAA and chitosan). The response of explants was observed and recorded based on the swelling, greenish color of the explants after one week from the inoculation. Similarly, shoot initiation and proliferation were recorded based on the size of the explants after two weeks and three weeks DPI (days post-inoculation) respectively. The multiplied explants were separated thoroughly and transferred to rooting medium i.e. half-strength MS, IBA and chitosan. The roots data scored based on the size of the roots. All roots data documentation after one week and two weeks from the inoculation.
       
The well-developed roots were washed thoroughly under the running tap water. The fungicide of Bavastin (1.5-2 g/L) was used for treating all rooted plantlets for twenty minutes. The roots plants were shifted into polythene bags which contain autoclaved sand, soil rite and cow excrete i.e. all were in one ratio and kept for 5-8 days with sprinkling water in room condition. Then all packing plantlets were carried out to the greenhouse. Finally, rooted plants were transferred to the field condition after one month.

Data analysis
 
The experiments were repeated three times and each treatment was ten explants. The data recorded on the response of explants, shoot initiation, shoot proliferation and formation of the roots in both explants. Characteristics of the explants were analyzed by ANOVA and means were compared using Duncan’s multiple range test (Gomez and Gomez, 1984) at a 5% significance level (p≤0.05).

The optimum ten different concentrations were selected out of 100 different concentrations for the response of both rhizome and sucker explants (Tables 1 and 2).
 

 

       
The maximum responses, shoot initiation and shoot proliferation were observed when a concentration as MS+5 mg/L BAP+0.5 mg/L IAA+25 mg/L chitosan in the rhizome and MS+4 mg/L BAP+0.5 mg/L IAA+25 mg/L chitosan in the sucker explants (Table 3 and Graph 1). The different developmental stages of rhizome and sucker plants were observed after one, two and three weeks respectively from the day post-inoculation (Fig 1 and Fig 2). According to the increases of 6-Benzylaminopurine concentration up-to 5.0 mg/L similarly increases of shoot proliferation and thereafter decreased but in both explants, as 0.5 mg/L IAA concentration remained constant. Previous researchers already reported that the percentageof shoot regeneration high with the increases of 6-Benzylaminopurine concentration up to 5.0 mg/l and thereafter declined (Sidha et al., 2007; Perez-Hernandez and Rosell-Garcia, 2008; Darvari et al., 2010; Sultan et al., 2011). Likewise, many researchers were depicted that number of shoot initiation and multiplication highest (Deo and Pradhan, 2017) where the concentration at 4 mg/L BAP along with 0.5 mg/L IAA. Muhammad et al., (2007) was found the identical result by 4 mg/L BAP+1 mg/L IAA concentration. Both Ahmed et al., (2014) and Habiba et al., (2002) were observed at 4 mg/L BAP+2 mg/L IAA concentration shown the highest multiple shoot formations. The chitosan has a growth promoter not only in the orchid tissue culture of the plants but also in the in vitro culture of the several species of the plants (Nge et al., 2006; Ohta et al., 1999; AitBarka et al., 2004 and Sopalun et al., 2010).
 

 

 

 

       
After the completed the multiplication stage to follow the rooting stage. Both the explants were used to test the 10 treatments along with positive control. The best concentrations as half-strength MS medium+1.0 mg/L IBA+25 mg/L chitosan in the rhizome and half-strength MS+0.8 mg/L IBA+25 mg/L chitosan in sucker were observed maximum roots formation (Table 4, Graph 2). There were observed that highest roots induction (3.33±0.37) and (3.66±0.37) in rhizome but (3.66±0.49) and (4.33±0.37) in sucker respectively (Fig 3).
 

 

 

       
In field conditions, the survival rates of plantlets were observed 90% in the sucker and 88% in rhizome along with control as 66% and 63% respectively (Graph 3). All in vitro regenerated plants successfully developed in hardening conditions (Fig 4).
 

 

The Murashige and Skoog medium supplemented with various plant growth regulators (PGRs) and chitosan can better perform for the regeneration of plantlets through in vitro culture of banana plants. However, this technique is financially cheaper and not time consumable. The rhizome and sucker as explants of bantala was the functional unit in the mass propagation of plantlets. So in this way, the demand of the market can be fulfilled. It’s likewise different cultivars development and adjusting requirement of the banana to the farmers.