Journal of Plant Biochemistry and Biotechnology

, Volume 22, Issue 4, pp 382–391 | Cite as

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

  • Diwakar Aggarwal
  • Neha Jaiswal
  • Anil Kumar
  • M. Sudhakara Reddy
Original article

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.

Keywords

Agrobacterium tumefaciens Carbenicillin RT-PCR 16S rRNA 

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

Notes

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.

References

  1. Aggarwal S, Kanwar K (2007) Comparison of genetic transformation in Morus alba L. via different regeneration systems. Plant Cell Rep 26:177–185CrossRefGoogle Scholar
  2. Aggarwal D, Kumar A, Reddy MS (2010) Shoot Organogenesis from elite plants of Eucalyptus tereticornis. Plant Cell Tissue Organ Cult 102:45–52CrossRefGoogle Scholar
  3. Aggarwal D, Kumar A, Reddy MS (2011) Agrobacterium tumefaciens mediated genetic transformation of selected elite clone(s) of Eucalyptus tereticornis. Acta Physiol Plant 33:1603–1611CrossRefGoogle Scholar
  4. Bakshi S, Sadhukhan A, Mishra S, Sahoo L (2011) Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration. Plant Cell Rep 30:2281–2292PubMedCrossRefGoogle Scholar
  5. Banarjee M, Srivastava S (2008) An improved protocol for in vitro multiplication of Bacopa monnieri (L.). World J Microbiol Biotechnol 24:1355–1359CrossRefGoogle Scholar
  6. Cai P, Long H, Deng GB, Pan ZF, Peng ZS, Yu MQ (2012) Molecular cloning, characterization, and expression analysis of genes encoding gibberellin 20-oxidase in Dasypyrum villosum dwarf mutant. Plant Mol Biol Rep. doi: 10.1007/s11105-012-0419-5
  7. Canter PH, Thomas H, Ernst E (2005) Bringing medicinal plants into cultivation: opportunities and challenges for biotechnology. Trends Biotechnol 23:180–185PubMedCrossRefGoogle Scholar
  8. Ceasar SA, Maxwell SL, Prasad KB, Karthigan M, Ignacimuthu S (2010) Highly efficient shoot regeneration of Bacopa monnieri (L.) using a two-stage culture procedure and assessment of genetic integrity of micropropagated plants by RAPD. Acta Physiol Plant 32:443–452CrossRefGoogle Scholar
  9. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  10. Dutt M, Li ZT, Dhekney SA, Gray DJ (2007) Transgenic plants from shoot apical meristems of Vitis vinifera L. “Thompson Seedless” via Agrobacterium-mediated transformation. Plant Cell Rep 26:2101–2110PubMedCrossRefGoogle Scholar
  11. Elangovan V, Govindasamy S, Ramamoorthy N, Balasubramanian K (1995) In vitro studies on the anticancer activity of Bacopa monnieri. Fitoterapia 66:211–215Google Scholar
  12. Godwin I, Todd G, Ford-Lloyd B, Newbury HJ (1991) The effect of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species. Plant Cell Rep 9:671–675CrossRefGoogle Scholar
  13. Gomez-Galera S, Pelacho AM, Gene A (2007) The genetic manipulation of medicinal and aromatic plants. Plant Cell Rep 26:1689–1715PubMedCrossRefGoogle Scholar
  14. Gurel S, Gulsen Y (1998) The effects of sucrose agar and pH levels on in vitro shoot production of Almond (Amygdalus communis L.). Turk J Bot 22:363–373Google Scholar
  15. Hammerschlag FA, Zimmerman RH, Yadava UL, Hunsucker S, Gercheva P (1997) Effect of antibiotics and exposure to an acidified medium on the elimination of Agrobacterium tumefaciens from apple leaf explants and on shoot regeneration. J Am Soc Hortic Sci 122:758–763Google Scholar
  16. Hoekema A, Hirsch PR, Schilperoort PJJ, Hooykaas RA (1983) A binary vector strategy based on separation of vir and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180CrossRefGoogle Scholar
  17. Holsters M, De Waele D, Depicker A, Messens E, Van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187PubMedCrossRefGoogle Scholar
  18. Hong YB, Liu SP, Zhu YP, Xie C, Jue DW, Chen M, Kaleri HA, Yang Q (2012) Expression of the MSI-99 m gene in transgenic potato plants confers resistance to Phytophthora infestans and Ralstonia solanacearum. Plant Mol Biol Rep. doi: 10.1007/s11105-012-0503-x
  19. James DJ, Uratsu S, Cheng J, Negri P, Viss P, Dandekar AM (1993) Acetosyringone and osmo-protectants like betaine or proline synergistically enhance Agrobacterium-mediated transformation of apple. Plant Cell Rep 12:559–563CrossRefGoogle Scholar
  20. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  21. Jung M, Shin SH, Park JM, Lee SN, Lee MY, Ryu KH, Paek KY, Harn CH (2011) Detection of transgene in early developmental stage by GFP monitoring enhances the efficiency of genetic transformation of pepper. Plant Biotechnol Rep 5:157–167PubMedCrossRefGoogle Scholar
  22. Krishnan VN, Soni KB, Rajmohan K (2008) Agrobacterium tumefaciens mediated genetic transformation in Centella asiatica L. Urban. Curr Biotica 2:1–8Google Scholar
  23. Kumar A, Sood A, Palni LMS, Gupta AK (1999) In vitro propagation of gladiolus hybridus hort.: synergistic effect of heat shock and sucrose on morphogenesis. Plant Cell Tissue Organ Cult 57:105–112CrossRefGoogle Scholar
  24. Kumar A, Palni LMS, Sood A, Sharma M, Palni UT, Gupta AK (2002) Heat shock induced somatic embryogenesis in callus cultures of gladiolus in the presence of high sucrose. J Hort Sci Biotechnol 77:3–78Google Scholar
  25. Kumar A, Chakraborty A, Ghanta S, Chattopadhyay S (2008) Agrobacterium-mediated genetic transformation of mint with E. coli glutathione synthetase gene. Plant Cell Tissue Organ Cult 96:117–126CrossRefGoogle Scholar
  26. Kumar A, Aggarwal D, Gupta P, Reddy MS (2010) Factors Effecting in vitro propagation and field establishment of Chlorophytum borivillianum. Biol Plant 54:601–606CrossRefGoogle Scholar
  27. Maheswaran GM, Welander M, Hutchinson JF, Graham MW, Richards D (1992) Transformation of apple rootstock M26 with Agrobacterium tumefaciens. J Plant Physiol 139:560–568CrossRefGoogle Scholar
  28. Mathur S, Gupta MM, Ram M, Sharma S, Kumar S (2002) Herb yield and bacoside A content of field grown Bacopa monnieri accessions. J Herb Spices Med Plants 9:11–18CrossRefGoogle Scholar
  29. Mehra PN (1972) Some aspects of differentiation in cryptogams. Res Bull (NS) Punjab University Part III–IV: 221–242Google Scholar
  30. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  31. Naik PM, Manohar SH, Praveen N, Murthy HN (2010) Effects of sucrose and pH levels on in vitro shoot regeneration from leaf explants of Bacopa monnieri and accumulation of bacoside A in regenerated shoots. Plant Cell Tissue Organ Cult 100:235–239CrossRefGoogle Scholar
  32. Nisha KK, Seetha K, Rajmohan K, Purushothama GM (2003) Agrobacterium-tumefaciens mediated transformation of Brahmi [Bacopa monnieri (L.)Wettst.], a popular medicinal herb of India. Curr Sci 85:85–89Google Scholar
  33. Padilla IMG, Burgos (2010) Aminoglycoside antibiotics: structure, functions and effects on in vitro plant culture and genetic transformation protocols. Plant Cell Rep 29:1203–1213PubMedCrossRefGoogle Scholar
  34. Padmanabhan P, Sahi SV (2009) Genetic transformation and regeneration of Sesbania drummondii using cotyledonary nodes. Plant Cell Rep 28:31–40PubMedCrossRefGoogle Scholar
  35. Pal R, Sarin JPS (1992) Quantitative determination of bacosides by UV spectrophotometry. Indian J Pharm Sci 54:17–18Google Scholar
  36. Pandey V, Misra P, Chaturvedi C, Mishra MK, Trivedi PK, Tuli R (2010) Agrobacterium tumefaciens-mediated transformation of Withania somnifera (L.) Dunal: an important medicinal plant. Plant Cell Rep 29:133–141PubMedCrossRefGoogle Scholar
  37. Pius J, George L, Eapen S, Rao PS (1993) Enhanced plant regeneration in pearl millet (Pennisetum americanum) by ethylene inhibitors and Cefotaxime. Plant Cell Tissue Organ Cult 32:91–96CrossRefGoogle Scholar
  38. Robert ML, Flores MR, Loyola-Vargas VM (1989) Growth promoting activity of certain penicillins on cultivated cells of Bouvardia ternifolia. Phytochem 28:2659–2662CrossRefGoogle Scholar
  39. Singh RH, Singh RL, Seni PO (1979) Studies on the anti-anxiety effect of the medha rasayana drug brahmi (Bacopa monniera). Part II experimental studies. J Res Ind Med Yoga Homeopath 14:1–6Google Scholar
  40. Sonia, Saini R, Singh RP, Jaiswal PK (2007) Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris α-amylase inhibitor-1 gene into mungbean: Vigna radiata (L.) Wilczek using bar as selectable marker. Plant Cell Rep 26:187–198PubMedCrossRefGoogle Scholar
  41. Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Identification of signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629CrossRefGoogle Scholar
  42. Tiwari V, Tiwari NK, Singh BD (1998) Shoot Regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monnieri (L.) Wettst.]. Plant Cell Rep 17:538–543CrossRefGoogle Scholar
  43. Tiwari V, Tiwari NK, Singh DB (2001) Comparative studies of cytokinins on in vitro propagation of Bacopa monnieri. Plant Cell Tissue Organ Cult 66:9–16CrossRefGoogle Scholar
  44. Tiwari V, Tewari KN, Singh BD (2006) Shoot bud regeneration from different explants of Bacopa monniera (L.) Wettst. by trimethoprim and bavistin. Plant Cell Rep 25:629–635PubMedCrossRefGoogle Scholar
  45. Tripathi YB, Chaurasia S, Tripathi E, Upadhaya A, Dubey GP (1996) Bacopa Monniera Linn as an antioxidant: mechanism of action. Ind J Exp Biol 34:521–526Google Scholar
  46. Volkov RA, Panchuk II, Schoffl F (2003) Heat-stress-dependency and developmental modulation of gene expression: the potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR. J Exp Bot 54:2343–2349PubMedCrossRefGoogle Scholar
  47. Wei XP, Gou XP, Yuan T, Russell SD (2006) A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica. Plant Cell Rep 25:513–521PubMedCrossRefGoogle Scholar
  48. Zuker A, Ahroni A, Tzfira T, Meir HB, Vainstein A (1999) Wounding by bombardment yields highly yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.). Mol Breeding 5:367–375CrossRefGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2012

Authors and Affiliations

  • Diwakar Aggarwal
    • 1
  • Neha Jaiswal
    • 1
  • Anil Kumar
    • 1
  • M. Sudhakara Reddy
    • 1
  1. 1.TIFAC- Centre of Relevance and Excellence in Agro and Industrial Biotechnology, Department of Biotechnology & Environmental SciencesThapar UniversityPatialaIndia

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