Controlling microbial contamination and browning of coconut (Cocos nucifera L.) inflorescence culture

Surface contamination of explants and culture browning are major problems associated with in vitro culture procedures. The study was aimed at investigating the effect of different sterilizing agents in controlling microbial contamination and browning in coconut ( Cocos nucifera L) inflorescence culture. Eeuwens medium, supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) at concentration 40 mg/l and 6-Benzyl amino purine (BAP) at concentration 1 mg/l was used for the experiment. Immature coconut inflorescences with intact inner spathes were used as explants. The explants were sterilized with different sterilizing agents: mercuric chloride (HgCl 2 ), sodium hypochlorite (NaClO), silver nitrate (AgNO 3 ) and calcium hypochlorite (Ca(ClO) 2 ), each at concentrations 0.1, 0.2, 0.3 and 0.4 % for 5 min sterilization time. This was followed by rinsing the explants with distilled water four successive times and were inoculated on Eeuwens medium. Percentage culture contamination and level of browning were recorded on weekly basis for four weeks. The results obtained showed that contamination was less in the cultures, whose explants were sterilized with HgCl 2 and AgNO 3 , but the level of culture browning was higher, four weeks after culture initiation when HgCl 2 was used for sterilization. By a way of comparison, AgNO 3 at concentrations between 0.1 - 0.3 % were most effective and to be preferred for sterilizing coconut inflorescence in controlling culture contamination and browning. It is therefore recommended that contamination and browning in coconut inflorescence cultures can be controlled by the use of AgNO 3 at concentrations between 0.1 – 0.3. Keywords:  Cocos nucifera , Immature inflorescence, Sterilizing agents, Contamination and Browning


INTRODUCTION
number of uses. The plant parts are used for In the tropical Pacific islands, coconut food, oil production, as construction material, source of energy, and cosmetics (Warner et al., (Cocos nucifera) palms play an important roles in 2007). Coconut cultivars are generally classified the culture, tourism, agriculture and into the Tall and Dwarf types. Most of the dwarf environment. Coconut is a palm that is grown all cultivars are used in genetic improvement over the world in about 12.2 million ha of land, of programs (Bourdeix et al., 2005). However, the which 88% of these are located in the Asia Pacific dwarf cultivar of coconut is faced with several region (Warth, 1933). There are more than 86 challenges from drastic production constraints, countries on four continents where coconut palm which include pests and diseases. is grown (Thangaraj and Muthuswani, 1990). In the tropics, the coconut plant has the largest In addition, several aging coconut plantations Nigerian Institute for Oil Palm Research (NIFOR), are now being uprooted in order to make way for Benin City. The explants were collected from the the planting of new once (Warner et al., 2007). leaf axil frond, as described by Rillo, (1989) for oil Therefore, these challenges have necessitated palm. After collection of explants, it was wrapped the need to implement efficient coconut in aluminum foil and then taken to the laboratory for analysis. germplasm via in vitro technique of tissue culture that allows germination and conversion into Preparation of Culture Medium plantlets in a controlled environment.
Many research works have been done in The basal medium used for this order to propagate Coconut from different types experiment was the formulation of Eeuwens of explants such as immature embryos, anthers, (Eeuwens, 1976). The medium contained 35g/l and tender leaves (Branton and Blank, 1984). In sucrose and 2.5 g/l activated charcoal and was the present study, immature inflorescence was supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) at concentration 40 mg/l and 6used as the explant for in vitro culture Benzyl amino purine (BAP) at concentration 1 establishment. One of the greatest problems mg/l for morphological responses. The pH of the associated with micropropagation procedure is medium was adjusted to 5.7 and solidified with 7 contamination with fungi and bacteria.
g/l agar. The medium was autoclaved at 121°C Therefore, an aseptic environments are essential for 20 min. for successful tissue culture procedures. To maintain a sterile condition, all culture vessels, Sterilization of Explants media and instruments used in handling tissues must be sterilized (Goswami and Handiqu, In order to investigate the effectiveness 2013). Sterilization is the process where by of the four different sterilizing agents in controlling microbial contamination, mercuric explants are made contamination free before in chloride, sodium hypochlorite, silver nitrate and vitro cultures are established. Several calcium hypochlorite, each at 0.1%-0.4% were sterilization agents are used to decontaminate used for sterilizing coconut inflorescence. The tissues during sterilization and should be carried explants were washed with detergent and rinsed out in such a way that the plant materials will not repeatedly with running tap water for 10-15 min, lose their biological activity. Therefore, explants before external spathes were removed under are surface sterilized only by treatment with aseptic conditions. The inflorescences with the disinfectant solution at suitable concentrations intact inner spathes were then introduced into for a specified period (Kumar et al., 2000). Apart the various sterilizing agents for 5 min, with from contamination, another major problem in in continuous stirring. The disinfectant solution vitro cultures of coconut inflorescence is was then discarded and the explants were rinsed phenolization. This term is described as the four successive times with sterile distilled water blackening and browning of explants shortly to avoid cytotoxic effect. after inoculation. It is caused by the release of phenolic compounds into the media where they Inoculation of Explants and Incubation of Cultures are immediately oxidized by peroxidases (El- The sterilized explants were inoculated Shafey et al., 1999). The objective of this study into the culture medium. The initiated cultures was to investigate the effectiveness of four o were incubated in the growth room at 25±2 C (in sterilizing agents (mercuric chloride, sodium the dark). Cultures were maintained in the dark hypochlorite, silver nitrate and calcium during the first 4 weeks for observations and hypochlorite) in controlling contamination and data on percentage contaminated culture and reduction of tissue browning of coconut in vitro.
level of culture browning were recorded on weekly basis for four weeks.

Source of Explants
Results Immature coconut inflorescences used The results obtained one week after for this study were obtained from the Dwarf culture initiation, when different sterilizing Green coconut palms in Anglican Church field, agents were used to sterilize coconut culture initiation, the results (   Table 3 presents the results obtained for culture browning increased. There was no culture microbial contamination and culture browning, three browning observed in cultures sterilized with 0.1% weeks after culture initiation. The level of culture AgNO . By the fourth week after culture initiation, only 3 contamination increased, but there was no cultures whose explants were sterilized with 0.1% contamination observed in cultures whose explants AgNO were observed not to have been contaminated 3 were sterilized with HgCl (0.4% concentration) and 2 and there was also no culture browning (Table 4). AgNO (0.1 and 0.4% concentrations), but the level of 3

Discussion
contamination rate when 1% silver nitrate was used to sterilize sour cherry shoot plant. Mercuric Media used for in vitro cultures also chloride also proved to be effective next to silver support growth of microorganisms, especially nitrate. For the first three weeks (Tables 1-3), bacteria and fungi, which have higher microbial contamination was not observed in the proliferating capability than plant cells, hence cultures who explants were sterilized with there is great need for surface sterilization of mercuric chloride, except in the fourth week, explants. The effectiveness of tissue sterilization where 6.7% contamination rate was recorded depends on certain factors which include explant ( Table 4). A similar observation by source, age, plant part and contamination levels Sushamakumari (2000) reported the use of of explants (Firoz et al., 2016). 0.1% -0.2% mercuric chloride for surface Microbial contamination (13.3%) was sterilization of immature inflorescence explants first observed in cultures whose explants were of Hevea. Other researchers such as Oyebanji et sterilized with sodium hypochlorite (0.1% concentration), four days after culture initiation.
al. (2009) have also reported sodium Contamination was later observed in some of the hypochlorite to be a very effective killer of other cultures within the first week. This bacteria and fungi, but in the present study, the observation was similar with Hadiuzzaman et al.
use of sodium hypochlorite was not effective for (2001), who reported that contamination was the surface sterilization of coconut inflorescence. observed in banana shoot tip culture, 5 days Apart from microbial contamination, after inoculation of explants. All the sterilizing another major problem in coconut tissue culture agents at various concentrations were able to is the browning of culture shortly after provide basal control measure of reducing inoculation. The results obtained in this study microbial contamination, one week after (Table 1 and 2) indicated that all the tested inoculation.
sterilizing agents were able to control browning In the present study, among the for the first two weeks of culture initiation. Slight sterilizing agents tested, 0.1% silver nitrate browning was observed after two weeks of seemed to be the best in controlling microbal culture initiation in cultures whose explants were contamination in coconut inflorescence cultures. sterilized with mercuric chloride (0.3% and 0.4% There was no any contamination recorded in concentrations), silver nitrate (0.4% conc.) and cultures whose explants were sterilized with calcium hypochlorite (0.4% conc.). Also, silver 0.1% silver nitrate throughout the study period nitrate (0.1 %) seemed to be most effective in (Tables 1-4). A similar observation of the controlling culture browning. A similar report has effectiveness of silver nitrate in reducing culture been made in solving the problem of browning microbial contamination was made by Osterc et on date palm inflorescence explants (El-Shafey al. (2004), who reported a reduction in culture et al., 1999). s Plate 1: Culture establishment using different sterilizing agents on coconut inflorescence explants. a: Immature coconut inflorescence explant; Cultures established four weeks post inoculation using: b) Silver nitrate; c) Mercury chloride; d) Calcium hypo chlorite) Sodium hypo chlorite, sterilizing agents