Standardization of Explant Surface Sterilization Technique for Micropropagation in Andrographis paniculata Nees. 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 by 0.1 % mercuric chloride (HgCl2) sterilization for two minutes proved to be optimum for maximum survival percentage in juvenile phase explants (shoot tip and nodal segment) whilst 70 % ethanol washing for 25 seconds followed by 0.1 % HgCl2 sterilization for two minutes proved to be optimum 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 and physiological stages of the same plant resulting in establishment of aseptic culture. Key words: Ethanol , mercuric chloride , shoot tip, nodal segment, juvenile phase, vegetative phase. 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 in

north-eastern India as ‘Maha-tita’, literally ‘king of bitters’ and it is also acknowledged as ‘Bhuineem’, since the plant, though much smaller in size, shows similar appearance and has bitter taste as that of Neem (Azadirachta indica). Since time immemorial, Andrographis was used as a wonder drug in traditional Siddha and Ayurvedic systems of medicine as well as in tribal medicine in India and in some other countries for multiple clinical applications. A study was carried out to standardize the protocol for micropropagation in Andrographis paniculata. The most decisive step in explant preparation while standardizing micropropagation techniques is that of keeping the explant alive overcoming the problems of conamination. The explant or the piece of the plant tissue to be cultured is often the major source of contamination. The contaminant may be on the surface of the explant, between the cells or within the plant cells. In order to surmount such tribulations 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 explants i.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 tap water. The explants were then prewashed with Tween 20 emulsifier (2-3 drops in 100 ml sterile distilled water) for one minute followed by rinsing three times in sterile distilled water. Prior to surface sterilization, antibrowning treatment was given to control phenol exudation from the cut end of the tissues. Before disinfection, the explants were washed with 70 % ethanol (v/v) for 10-25 seconds and surface sterilized with 0.1 % ( w/v) mercuric chloride (HgCl2 ) for 2-6 minutes. For better contact of the sterilant (0.1 % HgCl2 ) with the explants, they were stirred for few minutes while disinfesting. The surface sterilized explants were finally rinsed in sterile distilled water under laminar airflow chamber to remove all traces of sterilizing agent (1) and placed on a sterilized petridish covered with sterilized filter paper to remove excess moisture present on the surface of the explant. Data on contamination, mortality and survival percentage were recorded. The contamination percentage was calculated using the following formula, Contamination (% ) = Number of cultures contaminated X 100

Total number of cultures inoculated RESULTS AND DISCUSSION

Surfaces of plant carry a wide range of microbial contaminants. To avoid this source of infection, the explant tissues must be thoroughly surface sterilized before inoculating it on the nutrient medium. Explants treated with Tween 20, a wetting agent improved the disinfestation by acting as a surfactant thereby removing the surface contaminants like soil and dust. While trimming the explants, phenols ooze out from the cut tissues, resulting in explant browning. In

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 enhances the contact of the disinfectant (0.1 % HgCl2 ) efficiently (4). After rinsing in ethanol, the explants were left exposed until the alcohol evaporates (3). Generally, to disinfect the plant tissues various sterilizing agents have been used. Mercuric chloride was found to be a very effective sterilizing agent at 0.1 % concentration in Andrographis. The chlorine gas released from HgCl2 was very penetrating that it destroyed the microorganisms present in most tissues of the explant (2). It is also important to be cautious that a surface sterilant is also toxic to the explant tissues. Therefore concentration of the sterilizing agent and duration of the treatment should be optimum to minimize tissue mortality of the explants due to over sterilization. Table 2. Standardization of surface sterilization for vegetative phase explants in Andrographis paniculata Nees. Duration of Treatment exposure 70 % Alcohol (sec) T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 10 10 10 10 10 25 25 25 25 25 0.1% HgCl2 (mins) 1 2 3 4 5 1 2 3 4 5 60.00 40.00 30.00 20.00 25.00 25.00 5.00 10.00 10.00 10.00 20.00 30.00 40.00 40.00 30.00 60.00 85.00 70.00 60.00 50.00 20.00 30.00 30.00 40.00 45.00 15.00 10.00 20.00 30.00 40.00 40.00 40.00 30.00 20.00 15.00 55.00 10.00 15.00 20.00 30.00 10.00 20.00 25.00 30.00 15.00 20.00 80.00 25.00 80.00 70.00 45.00 40.00 45.00 50.00 70.00 25.00 10.00 20.00 15.00 10.00 % CON % SUR % MOR % CON % SUR % MOR Shoot tip Nodal segment

% CON - % Contamination, % SUR - % Survival, % MOR - % Mortality (Browning or blackening of explants). Statistically not analysed.

Incase of Andrographis paniculata, surface sterilization with 70 % ethanol washing for 10 seconds followed by 0.1 % HgCl2 sterilization for two minutes proved to be optimum for maximum survival percentage in juvenile phase explants (Table.1) whilst 70 % ethanol washing for 25 seconds followed by 0.1 % HgCl2 sterilization for two minutes proved to be optimum for vegetative phase explants (Table.2). The treatment duration varied with the physiological stage of the explant. The morphogentic growth pattern of the cell changes according to the physiological age of the plant material, resulting in direct correlation between the sensitivity of the tissues to the sterilizing agent and the ontogenic age of the explants. Juvenile phase explants consisted mostly of newly formed delicate tissues than the matured tissues of the vegetative phase explants. Thus juvenile phase explants require lesser time to surface disinfect than vegetative phase explants resulting in establishment of aseptic cultures.

REFERENCES 1. George, E.F. and Sherrington, P.D. 1984. Plant Propagation by Tissue Culture. Exogenetics Limited, England.

2. Hamill, S.D., Sharrock, S.L and Smith, M.K. 1993. Comparison of decontamination methods used in initiation of banana tissue cultures from field collected suckers. Plant cell tissue organ cult. 33: 343 –346.

3. Kao, K.N.. and Michayluk,M.R. 1980. Plant regenation from mesophyll protoplast of alfalfa.Z.Pflanzen physiol., 96 : 135 – 141.

4. Roberta H.Smith . 2005. Plant Tissue Culture: Techniques and Experiments.2/e.Elsevier publishers, New Delhi.