Genomic DNA methylation of juvenile and mature Acacia mangium micropropagated in vitro with reference to leaf morphology as a phase change marker

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Tree Physiology, 24:401–407

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Genomic DNA methylation of juvenile and mature Acacia mangium micropropagated in vitro with reference to leaf morphology as a phase change marker

Franc-Christophe Baurens (1), Joris Nicolleau (1, 2), Thierry Legavre (1), Jean-Luc Verdeil (1) and Olivier Monteuuis (2, 3)

1. CIRAD-Amis, Programme BIOTROP, TA 40/03, avenue Agropolis, 34398, Montpellier, Cedex 5, France / 2. CIRAD-Forêt, Programme Arbres et Plantations, TA 10/C, Baillarguet, 34398 Montpellier, Cedex 5, France / 3. Corresponding author (olivier.monteuuis@cirad.fr) / Received June 24, 2003; accepted September 9, 2003; published online February 2, 2004

Summary

Genomic DNA methylation was analyzed in Acacia mangium Willd. microshoots micropropagated in vitro from juvenile and mature explants, and in relation to leaf morphology of the microshoots, which is considered a phase change indicator. Based on high performance liquid chromatography (HPLC) analyses, we found more DNA methylation in microshoots exhibiting juvenile leaf morphology (22.4%) than in microshoots of the mature phyllode morphological type (20.7%), irrespective of the age of the source material. Overall, the degree of DNA methylation in A. mangium microshoots was consistent with values reported for other angiosperms. Complementary investigations based on methylation sensitive amplification polymorphism (MSAP) techniques established that, of 1204 fragments revealed by the different primer pairs used, 49 (i.e., 4.08%) were derived from C^5mCGG methylated sites. Three of these C^5mCGG sites were exclusive to the juvenile plant material, and three sites were exclusive to the mature source. No fragments were associated specifically with leaf morphology, rather than with plant age. Thus, although the two age classes could not be distinguished based on a quantitative HPLC measure of DNA methylation, qualitative differences existed, as demonstrated by the six age-specific markers identified by MSAP. The reliability of the MSAP data was confirmed on a larger sample of juvenile plant material, which suggested that the total of six methylation markers detected is probably an underestimation of the age-related differences in DNA methylation that may exist between juvenile and mature plant materials.

Keywords: age, foliar dimorphism, HPLC, maturational traits, 5-methyl-deoxycytidine, MSAP, tissue culture, tropical legume tree.