Azotobacter is a free-living, diazotrophic bacteria commonly inhabiting aquatic and terrestrial environments. Molecular methodologies have been optimized and adapted to overcome traditional techniques. The new methodologies allow rapid access to microbial diversity and facilitate the discovery of new groups of microorganisms. The aim of the research was to screen the Azotobacter from marine water by molecular techniques. However, the total heterotrophic bacterial population (64 ? 107 and 39 ? 107) and Azotobacter population (47 ? 102 and 30 ? 102) were studied. The morphological differences of the isolates were also recorded, such as pale white, round, pin head colonies for sample O1; large, pale white, flat, slimy colonies for sample J10 and small, glistening, water drops like mucoid colonies for sample F9. The isolates, when subjected to the molecular studies, recorded that the DNA bands produced by the Azotobacter isolates were similar to that of the standard strain (lane 1). The 16s rRNA of Azotobacter using genus-specific oligonucleotide primers showed that the amplicon of the isolates (lanes 2, 3 and 4) were clear but lighter compared to the standard lane 1 which was thick. Since all the lanes scored the amplicon size of 1,080 bp, all were confirmed to be the members of Azotobacter sp. The associative organisms were also identified as Klebsiella and Pseudomonas by insilico analysis.

Azotobacter species are gram negative, large (2-10 ? 1-2.5 µ), obligatory aerobic, pleomorphic, free-living diazotrophic bacteria commonly inhabiting aquatic and terrestrial environments. The older population of bacteria include encapsulated forms having enhanced resistance to heat, desiccation and adverse conditions. The cysts germinate under favorable conditions to yield vegetative cells. They have the highest respiratory rates known among living organisms and are able to grow on a wide variety of carbohydrates, alcohols and organic acids, in addition to being able to fix nitrogen (Lei et al., 1997). The growth of all organisms depends on the availability of mineral nutrients, and none is more important than nitrogen, which is required in large amounts being an essential component of proteins, nucleic acids and other cellular constituents. There is an abundant supply of nitrogen in the earth’s atmosphere, nearly 79% in the form of N2 gas. However, N2 is unavailable for use by most organisms because there is a triple bond between the two nitrogen atoms, making the molecule almost inert. In order to be used for growth, it must be ‘fixed’ (combined) in the form of ammonium (NH4 +) or nitrate (NO3 –) ions. So, nitrogen is often the limiting factor for growth and biomass production in all environments, where there is suitable climate and availability of water to support life (Deacon, 2003).

Nodal explants, Annona squamosa, Multiple shoots, BAP, Kinetin, IBA