Standardization of Explant Surface Sterilization Technique for Micropropagation in Andrographis paniculataNees. Haripriya.S and M.

Kannan Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore 3 ABSTRACT Surface sterilization with 70 % ethanol washing for 10 seconds followed by0.1 % mercuric chloride (HgCl2) sterilization for two minutes proved to be optimum for maximumsurvival percentage in juvenile phase explants (shoot tip and nodal segment) whilst 70 % ethanolwashing for 25 seconds followed by 0.1 % HgCl2 sterilization for two minutes proved to beoptimum for vegetative phase explants (shoot tip and nodal segment) of Andrographis paniculata.The tissue response of the explant varied with treatment duration depending on the type andphysiological stages of the same plant resulting in establishment of aseptic culture.

INTRODUCTION Andrographis paniculata Nees. (Acanthaceae), is an erect annual herb extremely bitter in taste in each and every part of the plant body. The plant is known innorth-eastern India as Mahatita, literally king of bitters and it is also acknowledged as Bhui-neem, since the plant, though much smaller in size, shows similar appearance and has bitter tasteas that of Neem (Azadirachta indica). Since time immemorial,Andrographis was used as a wonderdrug in traditional Siddha and Ayurvedic systems of medicine as well as in tribal medicine in Indiaand in some other countries for multiple clinical applications. A study was carried out tostandardize the protocol for micropropagation in Andrographis paniculata. The most decisive stepin explant preparation while standardizing micropropagation techniques is that of keeping theexplant alive overcoming the problems of conamination. The explant or the piece of the planttissue to be cultured is often the major source of contamination. The contaminant may be on thesurface of the explant, between the cells or within the plant cells. In order to surmount suchtribulations detrimental to the culture, the explants ought to be surface-sterilized before inoculation

in sterile growth medium. This paper details the research undertaken to standardize expalnt surface sterilization techniques for micropropagation in Andrographis paniculata Nees. MATERIALS AND METHODS The experiment was conducted at the Plant Tissue culture laboratory, HC & RI, TNAU,Coimbatore. The stock plant,Andrographis was maintained in pot culture at Botanic Gardens,TNAU, Coimbatore for supply of explantsi.e., shoot tip (1.5 - 2 cm) and nodal segment (2 -2.5 cm)throughout the experiment period. The explants were collected at two physiological stages of interest viz., juvenile phase (30-45 days old seedlings) and vegetative phase (60-90 days old plants) for micropropagation. Initially the freshly collected explants were washed thrice under running tapwater. The explants were then prewashed with Tween 20 emulsifier (2-3 drops in 100 ml steriledistilled water) for one minute followed by rinsing three times in sterile distilled water. Prior tosurface sterilization, antibrowning treatment was given to control phenol exudation from the cutend of the tissues. Before disinfection, the explants were washed with 70 % ethanol (v/v) for 10-25seconds and surface sterilized with 0.1 % ( w/v) mercuric chloride (HgCl2 ) for 2-6 minutes. Forbetter contact of the sterilant (0.1 % HgCl2 ) with the explants, they were stirred for few minuteswhile disinfesting. The surface sterilized explants were finally rinsed in sterile distilled waterunder laminar airflow chamber to remove all traces of sterilizing agent (1) and placed on asterilized petridish covered with sterilized filter paper to remove excess moisture present on thesurface of the explant. Data on contamination, mortality and survival percentage were recorded.The contamination percentage was calculated using the following formula, Number of cultures contaminated Contamination (% ) = X 100 Total number of cultures inoculated RESULTS AND DISCUSSION Surfaces of plant carry a wide range of microbial contaminants. To avoid this source ofinfection, the explant tissues must be thoroughly surface sterilized before inoculating it on thenutrient medium. Explants treated with Tween 20, a wetting agent improved the disinfestation byacting as a surfactant thereby removing the surface contaminants like soil and dust. Whiletrimming the explants, phenols ooze out from the cut tissues, resulting in explant browning. In<img

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order to control the phenol exudation, the explants were given antibrowning treatment. 70 %ethanol washing was given prior to disinfection, apart from a surface sterilant by itself, it enhancesthe contact of the disinfectant (0.1 % HgCl2 ) efficiently (4). After rinsing in ethanol, the explantswere left exposed until the alcohol evaporates (3). Generally, to disinfect the plant tissues varioussterilizing agents have been used. Mercuric chloride was found to be a very effective sterilizingagent at 0.1 % concentration inAndrographis. The chlorine gas released from HgCl2 was verypenetrating that it destroyed the microorganisms present in most tissues of the explant (2). It is alsoimportant to be cautious that a surface sterilant is also toxic to the explant tissues. Thereforeconcentration of the sterilizing agent and duration of the treatment should be optimum to minimizetissue mortality of the explants due to over sterilization. Table 2. Standardization of surface sterilization for vegetative phase explants in Andrographis paniculataNees. Duration of exposure Shoot tip Nodal segment Treatment 70 % Alcohol (sec) 0.1% HgCl2 (mins) % CON % SUR % MOR % CON % SUR % MOR T1 10 1 60.00

20.00 20.00 40.00 10.00 45.00 T2 10 2 40.00 30.00 30.00 40.00 20.00 40.00 T3 10 3 30.00 40.00 30.00 30.00 25.00 45.00 T4 10 4 20.00 40.00 40.00 20.00 30.00 50.00 T5 10 5 25.00 30.00 45.00 15.00 15.00 70.00 T6 25 1 25.00 60.00 15.00 55.00 20.00 25.00 T7 25 2 5.00 85.00

10.00 10.00 80.00 10.00 T8 25 3 10.00 70.00 20.00 15.00 25.00 20.00 T9 25 4 10.00 60.00 30.00 20.00 80.00 15.00 T10 25 5 10.00 50.00 40.00 30.00 70.00 1 0.00 % CON - % Contamination, % SUR - % Survival, % MOR - % Mortality (Browning or blackening of explants). Statistically not analysed.