Agrobacterium rhizogenes mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone treatment

Vinod Kumar
Plant Cell Biotechnology Department
Central Food Technological Research Institute
Mysore - 570 020, Karnataka State, India
Tel: 91 0821 2516 501
Fax: 91 0821 2517 233
E-mail: pcbt@cftri.res.in

Ashwani Sharma
Plant Cell Biotechnology Department
Central Food Technological Research Institute
Mysore - 570 020, Karnataka State, India
Tel: 91 0821 2516 501
Fax: 91 0821 2517 233
E-mail: pcbt@cftri.res.in

Bellur Chayapathy Narasimha Prasad
Plant Cell Biotechnology Department
Central Food Technological Research Institute
Mysore - 570 020, Karnataka State, India
Tel: 91 0821 2516 501
Fax: 91 0821 2517 233
E-mail: pcbt@cscftri.ren.nic.in

Harishchandra Bhaskar Gururaj
Plant Cell Biotechnology Department
Central Food Technological Research Institute
Mysore - 570 020, Karnataka State, India
Tel: 91 0821 2516 501
Fax: 91 0821 2517 233
E-mail: pcbt@cftri.res.in

Gokare Aswathanarayana Ravishankar*
Plant Cell Biotechnology Department
Central Food Technological Research Institute
Mysore - 570 020, Karnataka State, India
Tel: 91 0821 2516 501
Fax: 91 0821 2517 233
E-mail: pcbt@cftri.res.in

*Corresponding author

Financial support: Department of Biotechnology, Government of India for research grants and Council of Scientific and Industrial Research (CSIR) New Delhi for the award of research fellowships to VK, AS, BCNP and HBG.

Keywords: Agrobacterium rhizogenes, hairy roots, Nicotiana tabacum, transformation frequency, ultra-sonication.

The phytopathogenic bacteria Agrobacterium rhizogenes genetically transforms plants. Plant species differ in their temporal competence for transformation. Agrobacterium rhizogenes mediated transformation efficiency was assessed under the influence of sonication, calcium treatment, acetosyringone and macerating enzymes in suitable combinations in Nicotiana tabacum as a model system. Manual wounding resulted in 21% transformation frequency. Where as sonication resulted in 2.2 fold increase, followed by sonication with CaCl₂ treatment resulted in 2.5 fold increase and sonication with acetosyringone treatment resulted in 4.1 fold increase in transformation frequency. The results of this study may be very useful in genetic manipulation of plants by Agrobacterium rhizogenes mediated gene delivery to higher plants, which are recalcitrant to A. tumefaciens mediated genetic manipulation.

BIP Article

Various species of bacteria are capable of transferring genes to higher plant species (Broothaerts et al. 2005). Among them, most widely studied ones are Agrobacterium tumefaciens and Agrobacterium rhizogenes. The soil bacterium Agrobacterium rhizogenes infects the plant tissues and leads to the formation of adventitious roots or it is called as “hairy roots”. Plant species differ greatly in their susceptibility to infection by Agrobacterium rhizogenes, Agrobacterium tumefaciens and other bacterial species which are capable of gene transfer to plants (Broothaerts et al. 2005). There are many reports that suggest the successful use of A. rhizogenes harbouring binary vectors with desired gene constructs (Christey, 2001) for plant genetic transformation. A number of factors in Agrobacterium mediated transformation process can limit transformation of a particular plant. These include the genotype, wounding of plant tissue, synthesis of phenolic vir gene inducers released by the plant, bacterial attachment, T-DNA transfer into the plant cytoplasm, T-DNA nuclear translocation and T-DNA integration (Gelvin, 2000).

No reports available on influence of physical and chemical treatments on enhancement of transformation rates in A. rhizogenes mediated transformation. In this study we carried out extensive work on enhancement of transformation efficiency in Nicotiana tabacum as a model system and demonstrated that ultrasonication and acetosyringone treatment is highly effective for obtaining transgenics using A. rhizogenes.

Materials and Methods

Seeds of tobacco (Nicotiana tabacum var. Anand 115) were surface sterilized with 0.1% mercuric chloride cultured on MS basal media (Murashige and Skoog, 1962) without any phytohormone for in-vitro germination. Agrobacterium rhizogenes strain A4 was grown on LB medium and used for all the treatment.

The leaf explants of Nicotiana tabacum were infected by co-cultivation with Agrobacterium rhizogenes to induce hairy roots. Different physical and chemical parameters were assessed which could influence transformation frequency. All the explants were cultured on sterilized petriplates comprising semi solid MS (Murashige & Skoog 1962) medium without phytohormones, however supplemented with sucrose 30 g L^-1 and myo-inositol 100 mg L^-1. The medium was gelled with 0.8% (w/v) agar. The pH was adjusted to 5.8 ± 0.2. The medium was autoclaved under 1.06 kg/cm^-2 pressure at 121ºC for 15 min.

The leaf discs were pricked manually with metal needle (~10 wounds/cm²), dipped in Agrobacterium rhizogenes culture and incubated in a shaker at 70 rpm for 30 min in dark. The explants were blot dried using sterile filter paper and inoculated on co-cultivation medium.

The leaf segments were taken in a 50 ml polypropylene tube and ultra-sonicated at 50 MHz for 10-100 sec at 80% amplitude, dipped in Agrobacterium rhizogenes culture, incubated in a shaker at 70 rpm for 30 min in dark. The explants were blot dried using sterile filter paper and inoculated on co-cultivation medium.

The explants were sonicated, treated with cell wall degrading enzymes at different concentrations, washed thoroughly to remove traces of cellulase and pectinase enzyme. The explants after sonication and macerating enzyme treatments were dipped in Agrobacterium rhizogenes culture incubated in a shaker at 70 rpm for 30 min in dark. The explants were blot dried using sterile filter paper and inoculated on co-cultivation medium.

Acetosyringone 50-150 μM was incorporated in the co-cultivation medium. The sonicated explants dipped in A. rhizogenes culture, incubated in a shaker at 70 rpm for 30 minutes in dark. The explants were blot dried using sterile filter paper and inoculated on co-cultivation medium.

Five to 20 mM CaCl₂ was incorporated in co-cultivation medium. The sonicated explants dipped in Agrobacterium rhizogenes culture incubated in a shaker at 120 rpm for 30 min in dark. The explants were blot dried using sterile filter paper and inoculated on co-cultivation medium.

Hairy roots were made bacteria free by transferring to fresh medium containing the antibiotics. The transgenic nature of hairy roots were confirmed by Polymerase Chain Reaction (PCR) and southern hybridization.The results were expressed in percentage transformation frequency. All the experiments were carried out in triplicate and the results were expressed as mean + SD.

Results

Hairy roots were formed only from wounded regions. Each type of infection and wounding method showed unique pattern of hairy root induction with varying percentage of transformation frequency. Infection of leaf explants by manual wounding resulted in induction of hairy roots originating from the mid vein region. However, sonication treatment alone and with acetosyringone and calcium ion treatments resulted in induction of hairy roots from all over the surface of the leaf explants.

The transgenic nature of hairy roots was confirmed by PCR. A 308 bp rol A expected size fragments was obtained only in hairy roots and absent in normal roots. This amplicon reacted with the rol A specific probe in southern hybridization, confirmed the transgenic nature of the roots. Manual wounding resulted in 21% transformation frequency. Sonication treatment of leaf explants resulted in 2.2 fold increase in terms of transformation frequency when compared to manual wounding. Macerating enzyme treatment in combination with sonication resulted in reduced transformation frequency. Sonication assisted transformation resulted in 46% transformation frequency. Sonication with 5 mM CaCl₂ treatment in the co-cultivation medium resulted in 2.5 fold increase in transformation frequency. Whereas 60% transformation was obtained at 10 mM CaCl₂. Sonication with 100 µM acetosyringone treatment resulted in 4.1 fold increase with 86% transformation frequency. This treatment was found to be most efficient in enhancing the transformation frequency.

Discussion

Tobacco serves as an excellent model system to study the factors, which influence genetic transformation. Wounding is a prerequisite for the genetic transformation process. Acetosyringone is used successfully to enhance transformation in various plant species in A. tumefaciens mediated genetic transformation. The cell wall disruption caused by the lower energy ultrasonic frequency utilized in the present study is apparently very useful for Agrobacterium rhizogenes mediated transformation. Sonication has been used to enhance Agrobacterium tumefaciens mediated transformation of many different plant species (Trick and Finer, 1997). Acetosyringone has been known to enhance transformation efficiency due to activation of vir genes in A. tumefaciens (Gelvin, 2000). Therefore we presume that the enhancement in transformation by acetosyringone treatment may be due to activation of vir genes which is absolutely required for the T-DNA delivery to plant tissues.

We conclude that, wounding of host tissue by ultrasonication and treatment with acetosyringone followed by exposure to A. rhizogenes results in enhanced transformation frequency. This may be useful in transfer of genes to recalcitrant plants using A. rhizogenes.

References

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