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Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L.

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Abstract

An efficient method for Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L. is described. Embryogenic cell suspension cultures derived from stem internode callus were transformed with Agrobacterium tumefaciens harbouring pCAMBIA 1301 plant expression vector. Transformed colonies were selected on medium supplemented with hygromycin (5 mg/l). Continuously growing transformed cell suspension cultures were initiated from these colonies. Expression of β-glucuronidase in the suspension cultures was analysed by RT-PCR and GUS histochemical staining. GUS specific activity in the transformed suspension cultures was quantified using a MUG-based fluorometric assay. Expression levels of up to 105,870 pmol 4-MU/min/mg of total protein were noted in the transformed suspension cultures and 67,248 pmol 4-MU/min/mg of total protein in the spent media. Stability of GUS expression over a period of 7 months was studied. Plantlets were regenerated from the transformed embryogenic cells. Stable insertion of T-DNA into the host genome was confirmed by Southern blot analysis. This is the first report showing stable high-level expression of a foreign protein using embryogenic cell suspension cultures in S. album.

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Abbreviations

BAP:

Benzylaminopurine

2,4-D :

2,4-Dichlorophenoxy acetic acid

GA3 :

Gibberllic acid

IAA:

Indole-3-acetic acid

MUG:

4-Methylumbelliferyl-β-d-glucuronide

RT-PCR:

Reverse transcriptase-polymerase chain reaction

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Authors and Affiliations

  1. Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400 085, India

    U. K. S. Shekhawat, T. R. Ganapathi, L. Srinivas & V. A. Bapat

  2. Tree Improvement and Propagation Division, Institute of Wood Science and Technology, 18th Cross, Malleswaram, Bangalore, 560 003, India

    T. S. Rathore

Authors
  1. U. K. S. Shekhawat
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  2. T. R. Ganapathi
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  3. L. Srinivas
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  4. V. A. Bapat
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  5. T. S. Rathore
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Correspondence to T. R. Ganapathi.

Additional information

U. K. S. Shekhawat and T. R. Ganapathi contributed equally to this work.

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Shekhawat, U.K.S., Ganapathi, T.R., Srinivas, L. et al. Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L.. Plant Cell Tiss Organ Cult 92, 261–271 (2008). https://doi.org/10.1007/s11240-007-9330-4

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  • DOI: https://doi.org/10.1007/s11240-007-9330-4

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