Invitro Regeneration of Arabidopsis Thaliana from Suspension and Invitro Cultures and Analysis of Regeneration and Antioxidant Enzyme Levels
Arabidopsis tissue culture is valuable for any laboratory working on this model plant. Tissue culture methodology facilitates the production of a large number of plants that are genetically identical over a relatively short growth period. Currently this in vitro regeneration system is a good system to study the mechanism by which plants show regenerative plasticity. Plant regeneration is a key technology for successful stable plant transformation, while cell suspension cultures can be exploited for metabolite profiling, kinetic study and mining. In this paper we report methods for the successful and highly efficient in vitro regeneration of plants and production of stable cell suspension lines from cotyledons of Arabidopsis thaliana. It is an easy and reproducible method of regenerating Arabidopsis plants from callus culture. A combination of 6 benzylaminopurine BAP and a naphthalene acetic acid NAA in a Murashige and Skoog s MS based medium gives a high percentage of shoot formation. Further cell suspension culture were used to study the growth kinetics and also for checking level of antioxidant enzymes at different stages of culture. An expression analysis of antioxidant genes such as superoxide dismutase SOD and ascorbate peroxidase APX was also done at callus, shoot and root regeneration stage. It was found that levels and activity of these antioxidant enzymes were higher at regeneration stage, indicating antioxidant enzyme role in plant morphogenesis. Here we describe a standard protocol for regenerating Arabidopsis plants in tissue culture, and for preparing and observing samples using steriosome and bright field microscopy to study different stages of regeneration.
Arabidopsis thaliana, Suspension Culture, Callus Culture, Shoot Induction, Root Induction
Amrina Shafi | Insha Zahoor | Mudasir A Mir