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JXB Advance Access originally published online on March 31, 2003
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Journal of Experimental Botany, Vol. 54, No. 386, pp. 1361-1371, May 1, 2003
© 2003 Oxford University Press

Cytosine methylation occurs in a CDC48 homologue and a MADS-box gene during adventitious shoot induction in Petunia leaf explants1

Received 7 November 2002; Accepted 19 February 2003

A. Pavan Prakash6,2, Anil Kush3, Prakash Lakshmanan4 and Prakash P. Kumar5,2

2 Plant Morphogenesis Laboratory, Department of Biological Sciences, The National University of Singapore, 10 Science Drive 4, Singapore 117543
3 Reliance Life Sciences, Maker Chambers IV, 5th Floor, 222 Nariman Point, Mumbai 400 021, India
4 David North Plant Research Centre, BSES, PO Box 86, 50 Meiers Road, QLD 4068, Australia

Abbreviations: AzaC, 5-azacytidine; AzadC, 5-aza-2'-deoxycytidine; BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; CI, callus induction; COBRA, combined bisulphite restriction analysis; CRED-RA, coupled restriction enzyme digestion-random amplification; MS, Murashige and Skoog; NAA, {alpha}-naphthaleneacetic acid; ORF, open reading frame; SAM, shoot apical meristem; SI, shoot induction.

The DNA methylase inhibitors, 5-azacytidine and 5-aza-2'-deoxycytidine inhibited adventitious shoot induction in Petunia leaf cultures. Cytosine methylation status at CCGG sites in shoot- and callus-inducing culture treatments was analysed by coupled restriction enzyme digestion (HpaII or MspI) and random amplification. Two differentially methylated genomic DNA bands from the PCR products were cloned (OPU9-1 and OPU9-2) and sequenced. The open reading frames contained in OPU9-1 and OPU9-2 showed similarity to CDC48 and MADS-box genes, respectively. Cytosine methylation was restored at CCGG sites when the leaf explants were transferred from medium containing the drugs to medium without the drugs, simultaneously recovering the ability to develop adventitious shoot buds. Furthermore, combined bisulphite treatment and restriction analysis revealed differential methylation of CGCG sites in the drug-treated and control cultures. These results demonstrate that cytosine methylation at CCGG and CGCG sites within a MADS-box gene and a CDC48 homologue, among others, shows strong positive correlation with adventitious shoot bud induction in Petunia leaf explants.

Key words: CDC48 homologue, demethylating agents, DNA methylation, MADS-box gene, Petunia hybrida, shoot regeneration.


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