Stable lines of hairy roots were established from leaf explants of Bacopa monnieri using different strains (A4, R1000, SA79, MTCC 532 and MTCC 2364) of Agrobacterium rhizogenes. The efficiency of hairy roots induction of these strains varied significantly and the maximum transformation frequency (75 %) was observed in case of strain SA79 using leaf explants followed by internode (55 %) in the presence of acetosyringone. Different parameters such as cell density of Agrobacterium suspension, co-cultivation period and infection time influenced the root induction frequency. Maximum frequency of root induction was obtained with bacterial density of 0.6 OD 600 , 2 days of co-cultivation period and 10 min of infection time. Integration of T-DNA in the genome of hairy roots was confirmed by PCR amplification of rolB gene. Elimination of Agrobacterium from the established root cultures was ascertained by amplifying the DNA fragment specific to 16S rDNA and virD gene. All lines of hairy roots except strain A4 induced showed higher growth rate and accumulated higher levels of 'bacoside A' than the untransformed roots. Maximum biomass accumulation (6.8 g l -1 ) and 'bacoside A' content (10.02 mg g -1 DW) were recorded in case of the hairy root line induced by strain MTCC 2364.

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Bacoside A, a major active principle of Bacopa monnieri known for its cognitive effects is a mixture of saponins like bacoside A3, bacopaside II, isomer of bacopasaponin C and bacopasaponin C. Seasonal changes in biomass and bacoside A levels in fourteen accessions of B. monnieri were evaluated after maintaining these at a common site at Thapar University campus, Patiala (30°19 0 36.12 00 N and 76°24 0 1.08 00 E) for 1 year. Harvestable biomass and total bacoside A contents varied significantly between the accessions and also in a particular accession during different seasons of the year. The maximum dry weight of plant (biomass 1.64 g) and bacoside A levels (6.82 mg/plant) were recorded in accession BM1. Harvestable biomass was highest during summer in accessions BM1 and BM7 (FW 4.2 g/plant), whereas bacoside A levels were also highest during summer and in accession BM1 (6.82 mg/plant). The lowest bacoside A level (0.06 mg/plant) was recorded in accession BM14 during winter. Principal component analysis showed that samples of summer were positively correlated with both the components suggesting an appropriate time for the harvest.