Elimination and molecular identification of endophytic bacterial contaminants during in vitro propagation of Bambusa balcooa

Bambusa balcooa is an economically important, multipurpose bamboo species, decidedly used in construction industry. Availability of natural bamboo is depleting very rapidly due to accelerated deforestation and its unrestrained use. The large number and timely supply of saplings are the need of the hour for the restoration of bamboo stands. Micropropagation, being the potent alternative for season independent rapid regeneration, is restricted in bamboo because of endophytic contamination. An in vitro attempt has been taken to overcome the endophytic contamination by using broad spectrum antibiotics as surface sterilant as well as a media component. Ampicillin sodium salt (5 mg/ml for 30 min) as a surface sterilant was found as the best treatment for high bud breaking (80%) coupled with high branching and low contamination (20%) but it was found ineffective to control the contamination during multiplication stage. Then, two endophytes were isolated and minimum inhibitory concentration was determined through antibiotic susceptibility test for successful eradication at multiplication stage. Finally, contamination free cultures were obtained when streptocycline (100 μg/ml) and gentamicin sulphate (75 μg/ml) were added into the medium. The two isolated endophytes, BB1 and BB2, were identified through 16S rDNA techniques and NCBI-BLAST algorithm with 99% sequence similarity with those of Janibacter sp. (KX423734) and Serratia marcescens strain (KX423735). To our knowledge, this is the first report for B. balcooa where antibiotics were used as surface sterilant as well as medium component, to control endophytic bacterial contaminants, followed by their identification.