Skip to main content
SpringerLink
Log in

Effects associated with insertion of rol genes on morphogenic potential in explants derived from transgenic Bacopa monnieri (L.) Wettst

  • Original Article
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

The present study deals with the establishment of rolA-transgenic and rolB-transgenic plants for the first time through Agrobacterium tumefaciens mediated transformation, exploiting the inherent morphogenic potential of an important medicinal plant, Bacopa monnieri (L.) Wettst. The rolA-transgenic and rolB-transgenic plants showed integration and expression of rolA and rolB genes respectively, whereas Ri-transformed plants showed integration and expression of rolA, rolB, rolC and rolD genes. Morphogenic potential of different types of explants derived from rolA-transgenic, rolB-transgenic and Ri-transformed plants on basal medium was evaluated. Shoot organogenesis was enhanced significantly in leaf (1.6-fold) and internode (1.4-fold) explants derived from rolA-transgenic plants, rolB-transgenic leaf (2.4-fold) and internode (1.6-fold) explants as well as leaf (5.2-fold) and internode (3.3-fold) explants derived from Ri-transformed plants compared to explants from non-transformed plants. Substantial increase in root organogenesis was also noticed in rolA-transgenic leaf (1.7-fold) explants, rolB-transgenic leaf (3.6-fold) and internode (1.4-fold) explants as well as leaf (4.1-fold) and internode (1.9-fold) explants derived from Ri-transformed plants compared to non-transformed ones. In addition to this, growth of root tip and shoot regeneration was also noticed from Ri-transformed root explants, but not in rolA-transgenic, rolB-transgenic and non-transformed roots. Clones of each transgenic plant line harboring rol genes depicted notable phenotypic changes including reduced shoot and internode length, increased number of nodes/plant, leaves/plant and roots/plant. The leaf morphology was altered in rolB-transgenic and Ri-transformed plants but not in rolA-transgenic plants.

Key message

Insertion of rol genes, individual or in combination, of Agrobacterium rhizogenes resulted in enhanced morphogenic potential of excised explants and alteration in phenotype of transgenic Bacopa monnieri plants maintained in vitro.

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
Fig. 4

Similar content being viewed by others

References

  • Alpizar E, Dechamp E, Lapeyre-Montes F et al (2008) Agrobacterium rhizogenes-transformed roots of coffee (Coffea arabica): conditions for long-term proliferation, and morphological and molecular characterization. Ann Bot 101:929–940

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Altamura MM (2004) Agrobacterium rhizogenes rolB and rolD genes: regulation and involvement in plant development. Plant Cell Tiss Organ Cult 77:89–101

    Article  CAS  Google Scholar 

  • Altamura MM, D’Angeli S, Capitani F (1998) The protein of rolB gene enhances shoot formation in tobacco leaf explants and thin cell layers from plants in different physiological stages. J Exp Bot 49:1139–1146

    CAS  Google Scholar 

  • Bahramnejad B, Naji M, Bose R et al (2019) A critical review on use of Agrobacterium rhizogenes and their associated binary vectors for plant transformation. Biotechnol Adv 37:1–14

    Article  CAS  Google Scholar 

  • Beck E, Ludwig G, Averswald EA et al (1982) Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19:327–336

    Article  CAS  PubMed  Google Scholar 

  • Bellincampi D, Cardarelli M, Zaghi D et al (1996) Oligogalacturonides prevent rhizogenesis in rolB-transformed tobacco explants by inhibiting auxin-induced expression of the rolB gene. Plant Cell 8:477–487

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bettini PP, Marvasi M, Fani F et al (2016) Agrobacterium rhizogenes rolB gene affects photosynthesis and chlorophyll content in transgenic tomato (Solanum lycopersicum L.) plants. J Plant Physiol 204:27–35

    Article  CAS  PubMed  Google Scholar 

  • Bhojwani SS, Razdan MK (1983) Plant tissue culture: theory and practice. Amsterdam

  • Bulgakov VP, Vereshchagina YV, Bulgakov DV et al (2018) The rolB plant oncogene affects multiple signaling protein modules related to hormone signaling and plant defense. Sci Rep 8:2285

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Cardarelli M, Spano L, Mariotti D et al (1985) Identification of the genetic locus responsible for non polar root induction by Agrobacterium rhizogenes Ri plasmid. Plant Mol Bio 5:385–391

    Article  CAS  Google Scholar 

  • Casanova E, Zuker A, Trillas MI et al (2003) The rolC gene in carnation exhibits cytokinin- and auxin-like activities. Sci Hortic 97:321–331

    Article  CAS  Google Scholar 

  • Chaudhuri K, Das S, Bandyopadhyay M et al (2009) Transgenic mimicry of pathogen attack stimulates growth and secondary metabolite accumulation. Transgenic Res 18:121–134

    Article  CAS  PubMed  Google Scholar 

  • Chilton MD, Tepfer DA, Petit A et al (1982) Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells. Nature 295:432–434

    Article  CAS  Google Scholar 

  • Christensen B, Sriskandarajah S, Serek M et al (2008) Transformation of Kalanchoe blossfeldiana with rol-genes is useful in molecular breeding towards compact growth. Plant Cell Rep 27:1485–1495

    Article  CAS  PubMed  Google Scholar 

  • Delbarre A, Muller P, Imhoff V et al (1994) The rolB gene of Agrobacterium rhizogenes does not increase the auxin sensitivity of tobacco protoplasts by modifying the intracellular auxin concentration. Plant Physiol 105:563–569

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dellaporta SL, Woods J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21

    Article  CAS  Google Scholar 

  • Diouf D, Gherbi H, Prin Y et al (1995) Hairy root nodulation of Casuarina glauca: a system for the study of symbiotic gene expression in an actinorhizal tree. Mol Plant Microbe Interact 8:532–537

    Article  CAS  PubMed  Google Scholar 

  • Faiss M, Strnad M, Redig P et al (1996) Chemically induced expression of the rolC-encoded β-glucosidase in transgenic tobacco plants and analysis of cytokinin metabolism: rolC does not hydrolyze endogenous cytokinin glucosides in planta. Plant J 10:33–46

    Article  CAS  Google Scholar 

  • Hooykass PJJ, Klapwjik PM, Nuti MP et al (1977) Transfer of the A. tumefaciens Ti plasmid to avirulent Agrobacteria and Rhizobium ex planta. J Gen Microbiol 98:477–484

    Article  Google Scholar 

  • Kodahl N, Müller R, Lütken H (2016) The Agrobacterium rhizogenes oncogenes rolB and ORF13 increase formation of generative shoots and induce dwarfism in Arabidopsis thaliana (L.) Heynh. Plant Sci 252:22–29

  • Koul A, Sharma A, Gupta S et al (2014) Cost effective protocol for micropropagation of Bacopa monnieri using leaf explants. Int J Sci Res 3:210–212

    Google Scholar 

  • Largia MJV, Satish L, Johnsi R et al (2016) Analysis of propagation of Bacopa monnieri (L.) from hairy roots, elicitation and Bacoside A contents of Ri transformed plants. World J Microbiol Biotechnol 32:131–141

    Article  PubMed  CAS  Google Scholar 

  • Majumdar S, Garai S, Jha S (2011) Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants. Plant Cell Rep 30:941–954

    Article  CAS  PubMed  Google Scholar 

  • Majumdar S, Garai S, Jha S (2012) Use of the cryptogein gene to stimulate the accumulation of bacopa saponins in transgenic Bacopa monnieri plants. Plant Cell Rep 31:1899–1909

    Article  CAS  PubMed  Google Scholar 

  • Mauro ML, Costantino P, Bettini PP (2017) The never ending story of rol genes: a century after. Plant Cell Tiss Organ Cult 131:201–212

    Article  CAS  Google Scholar 

  • Mauro ML, Trovato M, Paolis AD et al (1996) The plant oncogene rolD stimulates flowering in transgenic tobacco plants. Dev Biol 180:693–700

    Article  CAS  PubMed  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor, New York

    Google Scholar 

  • Mukherjee A, Mazumder M, Jana J et al (2019) Enhancement of ABA sensitivity through conditional expression of the ARF10 gene in Brassica juncea reveals fertile plants with tolerance against Alternaria brassicicola. MPMI 32:1429–1447

    Article  CAS  PubMed  Google Scholar 

  • 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 

  • Owens LD, Cohen JD, Seelke R (1988) Gene introduction to induce morphogenesis. Hort Sci 23:520–525

    Google Scholar 

  • Paul P, Sarkar S, Jha S (2015) Effects associated with insertion of cryptogein gene utilizing Ri and Ti plasmids on morphology and secondary metabolites are stable in Bacopa monnieri-transformed plants grown in vitro and ex vitro. Plant Biotechnol Rep 9:231–245

    Article  Google Scholar 

  • Petit A, David C, Dahl GA et al (1983) Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation. Mol Gen Genet 190:204–214

    Article  CAS  Google Scholar 

  • Roychowdhury D, Majumder A, Jha S (2013) Agrobacterium rhizogenes-mediated transformation in medicinal plants: Prospects and challenges. In: Chandra S, Lata H, Varma A (eds) Biotechnology for medicinal plants: micropropagation and Improvement. Springer, Verlag, Berlin, pp 29–68

    Chapter  Google Scholar 

  • Saha PS, Sarkar S, Jeyasri R et al (2020) In vitro propagation, phytochemical and neuropharmacological profiles of Bacopa monnieri (L.)Wettst.: a review. Plants 9:411–435

    Article  CAS  PubMed Central  Google Scholar 

  • Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbour Press, Cold Spring Harbour, New York

    Google Scholar 

  • Sarkar S, Jha S (2017) Morpho-histological characterization and direct shoot organogenesis in two types of explants from Bacopa monnieri on unsupplemented basal medium. Plant Cell Tiss Organ Cult 130:435–441

    Article  CAS  Google Scholar 

  • Sarkar S, Ghosh I, Roychowdhury D et al (2018) The effects of rol genes of Agrobacterium rhizogenes on morphogenesis and secondary metabolite accumulation in medicinal plants. In: Kumar N et al (eds) Biotechnological approaches for medicinal and aromatic plants. Springer, Singapore, pp 27–51

    Chapter  Google Scholar 

  • Sevón N, Dräger B, Hiltunen R et al (1997) Characterization of transgenic plants derived from hairy roots of Hyoscyamus muticus. Plant Cell Rep 16:605–611

    Article  PubMed  Google Scholar 

  • Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol 11:118–131

    CAS  PubMed  Google Scholar 

  • Slightom JL, Durand-Tardif M, Jouanin L et al (1986) Nucleotide Sequence Analysis of TL-DNA of Agrobacterium rhizogenes agropine type plasmid. J Biol Chem 261:108–121

    Article  CAS  PubMed  Google Scholar 

  • Sokal RR, Rohlf FJ (1987) Introduction to biostatistics. WH Freeman, New York

    Google Scholar 

  • Tepfer D, Tempé J (1981) Production of d’agropine par des racines transformes sous I’action d’Agrobacterium rhizogenes souche A4. Comptes rendus de l’Académie des sciences 292:153–156

    CAS  Google Scholar 

  • Tran Than Van K (1980) Control of morphogenesis by inherent and exogenously applied factors in thin cell layers. Int Rev Cytol Suppl 11A:175–194

    Google Scholar 

  • van Altvorst AC, Bino RJ, van Dijk AJ et al (1992) Effects of the introduction of Agrobacterium rhizogenes rol genes on tomato plant and flower development. Plant Sci 83:77–85

    Article  Google Scholar 

  • Wabiko H, Minemura M (1996) Exogenous phytohormone-independent growth and regeneration of tobacco plants transgenic for the 6b gene of Agrobacterium tumefaciens AKE10. Plant Physiol 112:939–951

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang YM, Wang JB, Luo D et al (2001) Regeneration of plants from callus cultures of roots induced by Agrobacterium rhizogenes on Alhagi pseudoalhagi. Cell Res 11:279–284

    Article  CAS  PubMed  Google Scholar 

  • White FF, Taylor BH, Huffman GA et al (1985) Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J Bacteriol 164:33–44

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yadav S, Sharma P, Srivastava A et al (2014) Strain specific Agrobacterium-mediated genetic transformation of Bacopa monnieri. J Genet Eng Biotechnol 12:89–94

    Article  Google Scholar 

Download references

Acknowledgements

SS acknowledges Department of Biotechnology, GOI, for Senior Research Fellowship. S.J. is thankful to the National Academy of Sciences (NASI, Allahabad, India), for award of “Senior Scientist, NASI” and providing the financial support to continue the research. The authors thank the Head, Department of Botany, University of Calcutta for facilities provided.

Author information

Authors and Affiliations

  1. Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, West Bengal, 700 019, India

    Sayantika Sarkar & Sumita Jha

Authors
  1. Sayantika Sarkar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sumita Jha
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SS and SJ conceived and designed research. SS conducted this research, analyzed the results and wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Sumita Jha.

Ethics declarations

Conflict of interest

The authors declare no conflict of interests.

Additional information

Communicated by Ali R. Alan.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

11240_2021_2092_MOESM1_ESM.tif

Figure S1 Agarose gel electrophoresis showing expression of transgenes in different transgenic plant lines by RT-PCR. Supplementary material 1 (TIF 1713 kb)

11240_2021_2092_MOESM2_ESM.tif

Figure S2 a Comparative morphology of six-weeks-old in vitro grown different transgenic and non-transformed plants, b leaf morphology. Supplementary material 2 (TIF 1047 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sarkar, S., Jha, S. Effects associated with insertion of rol genes on morphogenic potential in explants derived from transgenic Bacopa monnieri (L.) Wettst. Plant Cell Tiss Organ Cult 146, 541–552 (2021). https://doi.org/10.1007/s11240-021-02092-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-021-02092-5

Keywords

Search

Navigation