Skip to main content

Advertisement

SpringerLink
Log in

Highly efficient production of transgenic Scoparia dulcis L. mediated by Agrobacterium tumefaciens: plant regeneration via shoot organogenesis

  • Original Article
  • Published:
Plant Biotechnology Reports Aims and scope Submit manuscript

Abstract

Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with β-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on the MS medium supplemented with 1 mg l−1 indole-3-butyric acid and 15 mg l−1 hygromycin. Successful transformation was confirmed by PCR analysis using uidA- and hpt-specific primers and monitored by histochemical assay for β-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at a rate of 54.6% with an average of 3.9 ± 0.39 transgenic plantlets per explant was achieved in the present transformation system. It took only 2–3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient and fast genetic transformation system was developed for S. dulcis using an Agrobacterium-mediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering studies in this medicinally important plant.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Abdin MZ (2007) Enhancing bioactive molecules in medicinal plants. In: Zhu Y, Tan B, Bay B, Liu C (eds) Natural products—essential resources for human survival. World Scientific, Singapore

    Google Scholar 

  • Ahsan M, Islam SK, Gray AI, Stimson WH (2003) Cytotoxic diterpenes from Scoparia dulcis. J Nat Prod 66:958–961

    Article  PubMed  CAS  Google Scholar 

  • Aileni M, Kokkirala VR, Kota SR, Umate P, Abbagani S (2008) Efficient in vitro regeneration from mature leaf explants of Scoparia dulcis L., an ethnomedicinal plant. J Herbs Spices Med Plants 14:200–207

    Article  CAS  Google Scholar 

  • An G (1985) High-efficiency transformation of cultured tobacco cells. Plant Physiol 79:568–570

    Article  PubMed  CAS  Google Scholar 

  • Babincová M, Schronerová K, Sourivong P (2008) Antiulcer activity of water extract of Scoparia dulcis. Fitoterapia 79:587–588

    Article  PubMed  Google Scholar 

  • Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363

    Article  PubMed  CAS  Google Scholar 

  • Bhattacharya R, Bhattacharya S (2001) High frequency in vitro propagation of Phyllanthus amarus Schum. & Thonn. by shoot tip culture. Indian J Exp Biol 39:1184–1187

    Google Scholar 

  • Bohnert H, Nguyen H, Lewis NG (2008) Bioengineering and molecular biology of plant pathways. Advances in plant biochemistry and molecular biology, vol 1. Elsevier, Amsterdam

    Google Scholar 

  • Chemler JA, Koffas MAG (2008) Metabolic engineering for plant natural product biosynthesis in microbes. Curr Opin Biotechnol 19:597–605

    Article  PubMed  CAS  Google Scholar 

  • Dixon RA (2005) Engineering of plant natural product pathways. Curr Opin Plant Biol 8:329–336

    Article  PubMed  CAS  Google Scholar 

  • Farnsworth NR, Soejarto DD (1991) Global importance of medicinal plants. In: Akerele O, Heywood V, Synge H (eds) Conservation and medicinal plants. Cambridge University Press, Cambridge, pp 25–51

    Chapter  Google Scholar 

  • Franke R, Michael CM, Meyer K, Shirley AM, Cusumano JC, Chapple C (2000) Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate 5-hydroxylase. Plant J 22:223–234

    Article  PubMed  CAS  Google Scholar 

  • Ghanti KS, Govindaraju B, Venugopal RB, Ramgopal RS, Kaviraj SP, Jabeen FTZ, Barad A, Rao S (2004) High frequency shoot regeneration form Phyllanthus amarus Schum. & Thonn. Indian J Biotechnol 3:103–107

    CAS  Google Scholar 

  • Grotewold E (2008) Transcription factors for predictive plant metabolic engineering: are we there yet? Curr Opin Biotechnol 19:138–144

    Article  PubMed  CAS  Google Scholar 

  • Hu Z, Wu YR, Li W, Gao H (2006) Factors effecting Agrobacterium tumefaciens-mediated gentic transformation of Lycium babarum L. In Vitro Cell Dev Biol Plant 42:461–466

    Article  CAS  Google Scholar 

  • James DJ, Uratsu S, Cheng J, Negri P, Viss P, Dandekar AM (1993) Acetosyringone and osmprotectants like betaine or proline synergistically enhance Agrobacterium-mediated transformation of apple. Plant Cell Rep 12:559–563

    Article  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) Gus fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  • Khan MY, Aliabbas S, Kumar V, Rajkumar S (2009) Recent advances in medicinal plant biotechnology. Indian J Biotechnol 8:9–22

    CAS  Google Scholar 

  • Kim SH, Hamada T (2005) Rapid and reliable method of extracting DNA form RNA form sweet potato [Ipomea batatas (L.) Lam.]. Biotechnol Lett 27:1841–1845

    Article  PubMed  CAS  Google Scholar 

  • Kirby J, Keasling JD (2009) Biosynthesis of plant isoprenoids: perspectives for microbial engineering. Annu Rev Plant Biol 60:35–355

    Article  Google Scholar 

  • Latha M, Pari L, Sitasawad S, Bhonde R (2004) Scoparia dulcis, a traditional antidiabetic plant, protects against streptozotocin induced oxidative stress and apoptosis in vitro and in vivo. J Biochem Mol Toxicol 18:261–272

    Article  PubMed  CAS  Google Scholar 

  • Latha M, Ramkumar KM, Pari L, Damodaran PN, Rajeshkannan V, Suresh T (2006) Phytochemical and antimicrobial study of an antidiabetic plant: Scoparia dulcis L. J Med Food 9:391–394

    Article  PubMed  CAS  Google Scholar 

  • Latha M, Pari L, Ramkumar KM, Rajaguru P, Suresh T, Dhanabal T, Sitasawad S, Bhonde R (2009) Antidiabetic effects of scoparic acid D isolated from Scoparia dulcis in rats with streptozotocin-induced diabetes. Nat Prod Res 14:1–13

    Google Scholar 

  • Mahmoud SS, Croteau RB (2001) Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase. Proc Natl Acad Sci USA 98:8915–8920

    Article  PubMed  CAS  Google Scholar 

  • Mann V, Harker M, Pecker I, Hirschberg J (2000) Metabolic engineering of astaxanthin production in tobacco flowers. Nat Biotechnol 18:888–892

    Article  PubMed  CAS  Google Scholar 

  • Mathew AJ, Jayachandran K (2009) Production of scopadulcic acid B from Scoparia dulcis Linn. using a luffa sponge bioreactor. Plant Cell Tissue Organ Cult 98:197–203

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Porika M, Aileni M, Gadidasu K, Kokkiral VR, Umate P, Rao AV, Devarakonda RK, Abbagani S (2009) In vitro HIV type 1 reverse transcriptase inhibitory acitivity of leaf extract of Scoparia dulcis L. J Herbs Spices Med Plants 15:241–247

    Article  Google Scholar 

  • Rashid H, Yokoi S, Toriyama K, Hinata K (1996) Transgenic plant production mediated by Agrobacterium in indica rice. Plant Cell Rep 20:701–705

    Google Scholar 

  • Riel MA, Kyle DE, Milhous WK (2002) Efficacy of scopadulcic acid A against Palsmodium falciparum in vitro. J Nat Prod 65:614–615

    Article  PubMed  CAS  Google Scholar 

  • Rosati C, Aquilani R, Dharmapuri S, Pallara P, Marusic C, Tavazza R, Bouvier F, Camara B, Giuliano G (2000) Metabolic engineering of beta-carotene and lycopene content in tomato fruit. Plant J 24:413–419

    Google Scholar 

  • Schäfer H, Wink M (2009) Medicinally important secondary metabolites in recombinant microorganisms or plants: progress in alkaloid biosynthesis. Biotechnol J 4:1684–1703

    Article  PubMed  Google Scholar 

  • Shimoda N, Toyoda-Yamamoto A, Nagamine J, Usami S, Katayama M, Sakagami Y, Michida Y (1990) Control of expression of Agrobacterium vir genes by synergistic action of phenolic signal molecules and monosaccharides. Proc Natl Acad Sci USA 87:6684–6688

    Article  PubMed  CAS  Google Scholar 

  • Taylor L (2006) Vassourinha monograph 4/Scoparia dulcis. In: The rainforest, pharmacy to the world. Raintree Nutrition, Inc., Carson City. http://www.rain-tree.net/Vassourinha-Monograph.pdf

  • Vander Fits L, Deakin EA, Hoge JH, Memelink J (2000) The ternary transformation system : constitive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation. Plant Mol Biol 43:495–502

    Article  CAS  Google Scholar 

  • Wang GL, Fang HL (1998) Mechanism and technology of plant genetic engineering. Science, Beijing

    Google Scholar 

  • Yamazaki M, Lin S, Hayashi T, Morita N, Asamizu T, Mourakoshi I, Saito K (1996) Transgenic fertile Scoparia dulcis L., a folk medicinal plant, conferred with a herbicide-restistant trait using an Ri binary vector. Plant Cell Rep 15:317–321

    Article  CAS  Google Scholar 

  • Ye X, Al-Babili S, Klöti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering of povitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287:303–305

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the National Basic Research Program (2007CB108805), the Shanghai Pujiang Talent Program (08PJ14109) and the CAS-TWAS Postgraduate Fellowship.

Author information

Authors and Affiliations

  1. National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China

    Mahender Aileni & Peng Zhang

  2. Plant Biotechnology Research Unit, Department of Biotechnology, Kakatiya University, Warangal, 506009, India

    Mahender Aileni & Sadanandam Abbagani

  3. Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China

    Peng Zhang

  4. Chenshan Botanical Garden, Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences, 3888 Chenhua Road, Shanghai, 201602, China

    Peng Zhang

Authors
  1. Mahender Aileni
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sadanandam Abbagani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peng Zhang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Aileni, M., Abbagani, S. & Zhang, P. Highly efficient production of transgenic Scoparia dulcis L. mediated by Agrobacterium tumefaciens: plant regeneration via shoot organogenesis. Plant Biotechnol Rep 5, 147–156 (2011). https://doi.org/10.1007/s11816-011-0166-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11816-011-0166-3

Keywords

Search

Navigation