Efficient Surface Sterilant for Establishment of Dendrobium in vitro Culture

Orchids are flowering plants, that belong to the orchidaceae family. Orchids are popularly grown for their wide range of colours i.e., red, pink, white, blue, green, purple, orange and yellow and a diverse variety with long vase life (Toukuhara and Mii, 2001). Orchidaceae is 2^nd largest family after Asteraceae. Some of the important genera are Bulbophyllum, Epidendrum, Dendrobium and Pleurothallis. Dendrobium is one of the important and 2^nd largest genera (Vendrame and Carvalho, 2008). Some of the species that are indigenous to India are D. aphyllum, D. transparens, D. densiflorum, D. fimbriatum and D. nobile.  It is perennial, has a non-woody structure and is epiphytic. Dendrobium nobile is a purple-coloured flower with a white colour at the center. It blooms in spring and winter. It is propagated vegetatively through keikis, shoot tip (Saiprasad and Polisetty, 2002), node (Pathania et al., 1998) and tissue culture. Tissue culture is becoming the most efficient and reliable method for the mass production of true to type healthy and disease-free plantlets (Martin et al., 2003; Çimen and Özge, 2009 and Raad et al., 2011).
       
The explants such as node, pseudobulb, leaf tips, etc. can be used for in vitro multiplication. It can multiply directly (organogenesis) or indirectly (callogenesis) by culturing the explants. Basal medium is supplemented through various plant growth regulators (such as auxin and cytokinin) at different concentrations which helps in the growth and development of the plantlet. Many problems were faced during the establishment such as phenolic oxidation (browning caused by the oxidation of phenolic compound), microbial contamination and minimum inoculation density.  For tissue culture operations to be successful, aseptic (free of all micro organisms) or sterile conditions must be maintained. Every culture vessel, medium and tool used in handling must be kept aseptic. Every procedure needs to be done in a sterile, laminar airflow cabinet (Chawala, 2003). The explant itself and its tissues need to be disinfected i.e., surface sterilization of explant. The main goal of disinfection techniques is to strike a balance between lowering infection and promoting explant survival and regeneration, both of which are greatly affected by the disinfectant employed and the physiological state of the explants (Traore et al., 2005); Jan et al., 2013). The source, size and age of explants are some of the components that affect the success of disinfection (Silva et al., 2016). Plant material so collected should be healthy and disease free. The size of the explants can affect the penetration of disinfectants and the efficiency of surface sterilization (Hall, 1999). In terms of the age of planting material, older plants may have dormant pathogens and therefore make disinfecting more challenging (Silva, 2013), Silva and Dobránszki, 2013). Along with this pretreatment of donor plants also plays a major role in reducing the cause of contamination (Silva et al., 2016). To eradicate the fungal or bacterial contamination, various methods and procedures have been developed (Hussain,1994; Herman, 1996). The rate of contamination can reduce the survival percentage, reduce multiplication and can lead to mortality. It is very challenging to achieve the planting material contamination free (Mihaljevic et al., 2013). Sterilization includes treatment with chemical solutions like mercuric chloride, sodium hypochlorite and ethanol (George, 1993). Since the plant tissues are also harmed by these sterilizing agents, the right concentration of sterilant, the length of time exposed to the sterilant and the application sequences of these sterilant must be standardized to reduce explant damage and improve survival (CPRI, 1992). Sterilant concentration can vary according to the planting material, species type, growth environment and age of the plant (Mihaljevic et al., 2013). Therefore, to control the problems of microbial contamination a protocol is necessary to be developed. Keeping this objective, the present investigation aims to establish an effective disinfection method for aseptic culture and increase the survival percentage of the explant of Dendrobium var. Sonia by using nodal segments.

The experiment was conducted at Tissue Culture Laboratory, BAC, Bihar Agricultural University (BAU Communication No. 1525/231003), Sabour, Bhagalpur, Bihar, India during the 2022-2023.
 
Collection of explants
 
The explants were collected from healthy plants of Dendrobium var. Sonia is grown and maintained at polyhouse complex, Bihar Agricultural University, Sabour. The orchid beds should be fumigated at least 24 hours before the collection of explant node and pseudobulb of age 1.5-2.0 months were selected as explant. Orchid plants were fumigated before 24 hours of explant collection. With the help of a sterile surgical blade, the explants were incised and collected in a sterile bottle. The explants were collected from healthy and disease free plants.
 
Cleaning and pre-treatment of explant
 
Collected explants were cleaned and incised into small segments (keeping at least one node in each segment) and kept in running water approximately for 30 minutes to remove all the adhering dust particles and then to sterile water wash. Then it was treated with fungicide (Mancozeb 2.0 g/l) and 2-3 drops of Tween-20 solution for 45 minutes with continuous agitation with the help of a shaker. Then it was given 2-3 sterile water wash followed by antibiotic treatment (0.1% Streptomycin sulphate) for 30 minutes and agitated continuously. And finally, rinse through sterile water (2-3 wash). Rest procedures were given in Laminar Air Flow Cabinet i.e., in a sterile condition.
 
Explant surface sterilization
 
The UV light of laminar air flow should be kept on for 45 minutes before use. The glassware (autoclaved) taken inside and the laminar air flow cabinet should be thoroughly wiped with 70% ethanol. The explants were rinsed with 2-3 wash of sterile water. There after, the explants were submerged in 70% ethanol for 5 minutes. Again given 3 sterile water wash. Followed by sterilant treatment. Sterilant viz. mercuric chloride (HgCl₂) and sodium hypochlorite (NaOCl) were used at different concentrations for varying time durations (Table 1). The explants were given a final sterile water wash for 3 times and now the explants were ready to be inoculated. The explants were incised in 1.0 to 1.5 cm onto autoclaved sheets with the help of a heat sterilized scalpel. Then the explants were inoculated in prepared MS media by holding the bottle near to burner (carrying out all the procedures near the burner). Further, the inoculated bottles were capped and sealed with polyethylene film and marking the date of inoculation on the top of the cap and kept the bottles in the incubation room in a controlled condition.
 

This investigation revealed that, significantly low percentage of contamination (30.40%) was observed in nodal segment explants, surface sterilized with the treatment T₂ (0.1% HgCl₂ for 10 minutes) followed by treatment T₆ (2% NaOCl for 20 minutes) with 32.80% contamination. Whereas, maximum contamination percent was observed in treatment T0 (autoclaved water). Contamination-free culture is an essential condition for successful in vitro regeneration. The growth of explants is dependent on the type of sterilant used, its concentration and for the time duration it is treated. The maximum part of the contamination was reduced from mancozeb treatment at 0.2%. In terms of the highest contamination-free explants (69.60%) were observed in treatment T₂ (0.1 % HgCl₂ for 10 minutes) with zero mortality followed by treatment T₆ i.e., 2% NaOCl for 20 minutes (67.20%) and lowest contaminated free culture was obtained at autoclaved water (Table 2). Significantly maximum aseptic culture (69.60%) was found in T₂ (0.1% HgCl₂ for 10 minutes) followed by treatment T₆ i.e., 2% NaOCl for 20 minutes (67.20). All cultures were found contaminated at treatment T₀ i.e., untreated control (Fig 1).
 

 

       
Contamination free culture is an essential condition for successful in vitro regeneration. The growth of explants is dependent on the type of sterilant used, their concentration and the time duration it is treated. The maximum part of the contamination was reduced from fungicide treatment i.e., to 0.2% Mancozeb and further reduced from sterilant used and the best result came out to be at 0.1% HgCl₂ at 10 minutes. Similar results were obtained, where 80% of survivability was obtained at 0.1% of HgCl₂ for 10 minutes and 70% ethanol (Parvaty, 2022). Maximum culture establishment (85.67%) was observed when explants were sterilized with 0.1% HgCl₂ for 3 minutes+ 70% C₂H₆O for 30 seconds in Anthurium (Anthurium andraeanum Lind.) cv. Jewel by Thokchom and Maitra, (2016). In Gerbera jamesonii cv. Rosalin, maximum survivability (64.89%) was found at the combination of 0.1% HgCl₂ for 1 minute and ethyl alcohol (70%) for 30 seconds by Thokchom and Maitra (2017). Ahamed and Ferdaous (2019) found 40% contamination free explants on 0.1% HgCl₂ for 6 minutes in Gerbera jamesonii Bolus. Whereas, Balilashaki et al., (2014) observed only 10% contamination at 7% sodium hypochlorite for 10 minutes in Phalaenopsis amabilis cv. Cool ‘Breeze’. Abubakar and Pudake (2019) reported the result of sterilization procedures, revealed 15% NaHClO₃ (5 min) + 70% ethanol (30s) + 0.1% HgCl₂ (5min) to be the most effective in controlling contamination in Curcuma caesia among all the treatments studied, with 85% contaminants free cultures after 16 days of inoculation. Maximum mortality was observed at treatment T₆ i.e., 2% NaOCl for 20 minutes (20.80) which resulted in 46.40% of culture establishment. However, the least mortality (0%) was observed at 0.1% HgCl₂ at 5 and 10 minutes. A reduced mortality percentage (11.33), with the least contamination percent (3.67) at 0.1% HgCl₂ for 3 minutes + 70% C₂H₆O for 30 seconds observed by Thokchom and Maitra (2016). Sharma and Beura (2021) observed 6% mortality at 0.1% HgCl₂ solution for 4-7 minutes followed by 0.5% NaOCl solutions for 2 minutes. Maximum culture establishment or survival percent (69.60) was obtained at 0.1% of HgCl₂ for 10 minutes. Maximum aseptic culture per cent (96.67) with maximum survivability (96.67%) in Phalaenopsis hybrids cv. Shagan was also reported by Madhuri et al., (2021). The seed treated with 0.1% HgCl₂ within 6 to 15 minutes showed that the survival rate increased from 34.29% to 75% in Red Lotus (Trang et al., 2021). In shoot culture of Dendrobium aurantiacum maximum sterilization efficiency was found at 20% NaOCl for 15 minutes which gives a 73.3±5.8% survival rate after 1 month of culture by Ma et al., (2020).

Overcoming the problem of poor sterilization effect of Dendrobium orchid caused by prolonged field-planting time and abundant phenolic substances in plants found in previous preliminary experiments, the aseptic system of node segments of Dendrobium var. Sonia was established, which is helpful to the efficient propagation of excellent individual plants. The successful establishment of the aseptic system is the basis for in vitro regeneration of plants, which provides a guarantee for successful tissue culture of Dendrobium. From the present investigation, it can be concluded that surface sterilization is a very crucial step for the maximum rate of survivability or establishment of culture. And best and desired results in terms of minimum contamination with maximum establishment can be obtained when the explants are treated with 0.1% HgCl₂ for 10 minutes.

The authors declare that they have no competing financial interests or personal relationship that could have appeared to influence the work reported in this paper.