Variation in oil palm (Elaeis guineensis Jacq.) tissue culture-derived regenerants revealed by AFLPs with methylation-sensitive enzymes

Tissue culture-derived plants of oil palm (Elaeis guineensis Jacq.) can dev elop abnormal flowers in which stamen primordia are converted into carpel-l ike tissues (mantled fruit). This abnormality can be heritable; individual palms may show variation in mantling and reversion to the normal phenotype over time has been observed. Four sets of ortets (mother plant used as tiss ue source) and ramets (regenerated plants) were compared using standard amp lified fragment length polymorphism (AFLP) analysis and AFLPs using methyla tion-sensitive enzymes. No polymorphisms were found when standard AFLPs wer e produced with ten different primer combinations. In contrast, when methyl ation-sensitive AFLPs were used, polymorphisms were detectable. Polymer phi sms appeared as new bands in the ramets, suggesting that a reduction in met hylation had occurred during tissue culture. The highest number of polymorp hic bands (0.3%) was obtained when HpaII was used as the restriction endonu clease, indicating that the loss of methylation had occurred most frequentl y at the internal C within the HpaII recognition sequence 5 ' -CCGG-3 '. Co nversion of nine of the polymorphic bands into probes for Southern analysis confirmed that these were not due to partial digestion of the AFLP templat es and showed that the majority were single-copy sequences. The exceptions were fragments showing homology to 25S ribosomal RNA genes and the chalcone synthase gene family. Examination of the Southern blots suggested that mos t of the single-copy sequences were partially de-methylated, and one exampl e was found in which de-methylation affected only one allele. No polymorphi sm was consistently different between normal and abnormal clones in all the sets. This suggests that, whilst this method is an effective way of detect ing variation in tissue culture-derived plants, different approaches will b e required to identify the causal basis of the mantled fruit abnormality.