Techniques In Molecular Biology And Plant Tissue Culture
Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Preparation of plant tissue for tissue culture is performed under aseptic conditions. Thereafter, the tissue is grown in sterile containers, such as petri dishes or flasks in a growth room with controlled temperature and light intensity. SH3a medium was prepared for tissue culture of chickpea. This nutrient medium provides nutrition to the developing explant and contains N6-Major salts, SH- Minor salts, Vitamins, EDFS, Sucrose, 2,4-D(an Auxin) and Phytagel as the solidifying substance. For the establishment of undifferentiated callus culture, the sterilized leaf sheath explants were inoculated on phytagel-solidified basal SH3a medium. Cultures were incubated in the controlled conditions of temperature (25±2°C) and light intensity (2000-2500 lux for 16 hours); the experiment was performed, observed regularly till 16 days and callus was reported. Under molecular biology techniques, genomic DNA of chickpea was isolated with CTAB (Cetyl Trimethyl Ammonium Bromide) buffer and Spectroscopy based quantification of genomic DNA was done. A gene fragment was amplified by PCR (polymerase chain reaction) of template DNA and its ligation with PGEMT vector was performed for cloning. E. coli strain DH5α was transformed with the ligated product and plasmid DNA isolated from the ampicillin-resistant colonies were checked with PCR for confirmation of cloning. In the upcoming weeks, I will be performing further processes involved in cloning and plant tissue culture.
Keywords: PCR, Cloning, Plant Tissue Culture, Plasmid DNA