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Materials and Methods
A. Surface sterilization of the seeds
B. Aseptic techniques
C. Tissue harvest and start of culture
The experiment of plant tissue culture was conducted with the aseptic techniques in the laminar flow hood to prevent contamination. The most important part of a hood is a high efficiency bacteria-retentive filter (Akers 1991). Room air is taken into the unit and passed through a pre-filter to remove gross contaminants such as lint and dust (Figure 1). The air is then compressed and channeled up behind and through the HEPA filter (High Efficiency Particulate Air filter) in a laminar flow fashion that is the purified air flows out over the entire work surface in parallel lines at a uniform velocity (Figure 1) (Akers 1991). The HEPA filter removes nearly all of the bacteria from the air (Akers 1991).
The previous study showed that the cultured plant required a source of energy from sugar, salts, a few vitamins, amino acids, etc. that are provided in the nutrient media (Thakur 2005). Petioles were surface-sterilized and then cultured in flasks containing MS medium supplemented with various combinations and concentrations of plant growth regulators such as BAP and NAA, BAP and IAA, Kn and NAA respectively (Thakur 2005). The regenerated shoots were separated and individual shoots were transferred to the MS medium containing various concentrations of different auxins, IAA and IBA, for root induction and elongation to get a complete plantlet (Thakur 2005).
Akers, M. J., G. E. Wright, and K. A. Carlson. 1991. Sterility Testing of Antimicrobial-
containing Injectable Solutions Prepared in the Pharmacy. American Journal of Hospital Pharmacy 48: 2414-2417.
ÖKTEM, H. A., Y. BÜLBÜL, E. ÖKTEM, and M. YÜCEL. 1999. Regeneration and
Agrobacterium-mediated Transformation Studies in Tomato. Department of Biology, Middle East Technical University. 23:345-348.
Thakur, A. K., S. Sharma, and D. K. Srivastava. 2005. Plant Regeneration and Genetic
Transformation Studies in Petiole Tissue of Himalayan Popular. Current Science 89: 664-669.
Zieslin, N., F. Starkman, and E. Zamski. Growth of Rose Flower Peduncles and Effects of
Applied Plant Growth Regulators. 2004. Plant Growth Regulation 10: 65-76.