Influence of Agrobacterium rhizogenes strains on hairy root induction and ‘bacoside A’ production from Bacopa monnieri (L.) Wettst.
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 OD600, 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.
KeywordsAgrobacterium rhizogenes Bacopa monnieri Hairy roots Bacoside A PCR
Authors are thankful to University Grant Commission (UGC), Govt. of India, New Delhi for the financial assistance. Professor A. K. Srivastava, Indian Institute of Technology, New Delhi is thanked for providing some of the strains of Agrobacterium rhizogenes and Professor Desh Bir Sharma is thanked for editing the English of the ms. Thanks are also due to TIFAC-CORE, Thapar University Patiala for the facilities to carry out this work.
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