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Factors affecting genetic transformation and shoot organogenesis of Bacopa monnieri (L.) Wettst

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Abstract

Efficient Agrobacterium tumefaciens mediated T-DNA delivery and subsequent shoot organogenesis has been achieved from Bacopa monnieri. Various factors influenced T-DNA delivery as evident from transient GUS assay. The transient GUS expression was significantly higher (97.7 %) in explants that were pre-cultured before bacterial infection on medium supplemented with 100 μM acetosyringone. Incorporation of acetosyringone into the co-cultivation medium also enhanced transient GUS activity. Explant injury with carborundum paper, co-cultivation period of 2 days and a bacterial density of 0.4 OD600 showed higher transient GUS expression. Following co-cultivation, shoot organogenesis was achieved from leaf segments on basal Murashige and Skoog medium containing 58 mM sucrose. Supplementation of antibiotics (cefotaxime or carbenicillin) at > 250 μg/ml into the medium significantly promoted shoot organogenesis from leaf explants (71.5 % in control and > 83.0 % on medium containing 500 μg/ml of carbenicillin or cefotaxime). Stable transformation of regenerated shoots was confirmed on the basis of GUS activity and PCR amplification of DNA fragments specific to reporter gene (uidA) and selection marker gene (nptII). The expression level of nptII gene in independent transgenic lines was studied using quantitative real time-PCR. Stable transformed shoots after rooting were successfully established in the pots.

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Abbreviations

GUS:

β-Glucuronidase

nptII :

Neomycin phosphotransferase

RT-PCR:

Reverse transcriptase-polymerase chain reaction

qRT-PCR:

Real Time - polymerase chain reaction

X-Gluc:

5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid

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Acknowledgments

Authors are thankful to University Grant Commission (UGC), Govt. of India, New Delhi and Thapar University, Patiala for the financial assistance. Thanks are also due to TIFAC-CORE, Thapar University Patiala for the facilities to carry out this work.

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Authors and Affiliations

  1. TIFAC- Centre of Relevance and Excellence in Agro and Industrial Biotechnology, Department of Biotechnology & Environmental Sciences, Thapar University, Patiala, 147004, India

    Diwakar Aggarwal, Neha Jaiswal, Anil Kumar & M. Sudhakara Reddy

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  1. Diwakar Aggarwal
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  2. Neha Jaiswal
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  3. Anil Kumar
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  4. M. Sudhakara Reddy
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Correspondence to Anil Kumar.

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Aggarwal, D., Jaiswal, N., Kumar, A. et al. Factors affecting genetic transformation and shoot organogenesis of Bacopa monnieri (L.) Wettst. J. Plant Biochem. Biotechnol. 22, 382–391 (2013). https://doi.org/10.1007/s13562-012-0166-6

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  • DOI: https://doi.org/10.1007/s13562-012-0166-6

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