BackgroundThe world’s top three cereals, based on their monetary value, are rice, wheat, and corn. In cereal crops, DNA extraction is difficult owing to rigid non-cellulose components in the cell wall of leaves and high starch and protein content in grains. The advanced techniques in molecular biology require pure and quick extraction of DNA. The majority of existing DNA extraction methods rely on long incubation and multiple precipitations or commercially available kits to produce contaminant-free high molecular weight DNA.ResultsIn this study, we compared three different methods used for the isolation of high-quality genomic DNA from the grains of cereal crop, Zea mays, with minor modifications. The DNA from the grains of two maize hybrids, M10 and M321, was extracted using extraction methods DNeasy Qiagen Plant Mini Kit, CTAB-method (with/without 1% PVP) and modified Mericon extraction. Genes coding for 45S ribosomal RNA are organized in tandem arrays of up to several thousand copies and contain codes for 18S, 5.8S and 26S rRNA units separated by internal transcribed spacers ITS1 and ITS2. While the rRNA units are evolutionary conserved, ITS regions show high level of interspecific divergence and have been used frequently in genetic diversity and phylogenetic studies. In this study, the genomic DNA was then amplified with PCR using primers specific for ITS gene. PCR products were then visualized on agarose gel.ConclusionThe modified Mericon extraction method was found to be the most efficient DNA extraction method, capable to provide high DNA yields with better quality, affordable cost and less time.

Heartwood formation is generally characterized by the accumulation of phenolic substances that increase the natural color and durability of wood. Although there is evidence that these substances are synthesized in aging sapwood cells, little is known about heartwood formation at the molecular level. We monitored seasonal changes in flavanol concentration across the stems of 23-year-old Juglans nigra L. trees by sampling growth rings extending from the differentiating xylem to the heartwood. We also analyzed expression of phenylpropanoid and flavonoid structural genes in these samples. In the sapwood-heartwood transition zone, flavanol accumulation was correlated with the transcription levels of the chalcone synthase (CHS) and flavanone 3-hydroxylase (F3H) genes. We also observed correlations between flavanol accumulation and the amount of dihydroflavonol 4-reductase (DFR) gene transcript in October, January and May. Although transcription of phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL) genes did not correlate with flavanol accumulation, PAL genes were strongly expressed in the transition zone of samples collected in autumn, suggesting that their transcription in these tissues contributes to phenolic biosynthesis. Western immunoblotting showed that accumulation of CHS protein correlated with the amount of CHS gene transcript, whereas accumulation of PAL protein did not correlate with the the transcription levels PAL genes. Preliminary analyses revealed that PAL and CHS activities were higher in the transition zone than in the inner sapwood in autumn, winter, and spring. Thus, CHS activity could be regulated mainly at the transcriptional level, whereas post-translational modifications could modulate PAL activity. We conclude that flavanols are synthesized de novo in J. nigra sapwood cells that are undergoing transformation to heartwood.

Summary In trunks of Juglans nigra and the hybrid J. major × J. regia, the presence of non-structural carbohydrates, sucrose synthesizing and degrading enzymes, and their correlation with heartwood formation was investigated. Contents of starch and sucrose were highest in the youngest sapwood, decreased with increasing age of the tissue, and were absent in the heartwood. Pools of the monosaccharides glucose and fructose were low in the sapwood, and fructose was absent from the heartwood. Glucose transiently increased at the sapwood heartwood boundary in trunks of the hybrid. In black walnut stems, however, glucose started to accumulate within the transition zone and reached considerable amounts in the heartwood. Cold-adaptation in walnut wood was characterized by accumulation of soluble sugars. Sucrose formation was enabled by enhanced rates of sucrose-phosphate synthase (SPS, EC 2.4.1.14). Mid-winter starch-sugar interconversion was accompanied by increases in the activity of sucrose synthase (SuSy, EC 2.4.1.13; black walnut), or acid invertases (EC 3.2.1.26; hybrid). In the tissues undergoing heartwood formation, sucrose breakdown was enhanced from late summer until early winter. Sucrolysis was dominated by acid invertases with minor contribution of sucrose synthase. The catalytic activity of UDP-glucose pyrophosphorylase (EC 2.7.7.9), involved in the metabolization of the sucrose cleavage products, followed this seasonal trend and showed elevated activities from late summer until early winter. These data are further proof for the earlier made hypothesis (Hauch and Magel 1998) that the in situ synthesis of heartwood flavonoids relies on an interaction between primary (sucrose) and secondary metabolism. Flavonoids, however, constitute only a minor fraction in the heartwood of walnut and the bulk of heartwood phenolics seem to derive from transformation of phenolic precursors. Therefore, these recent findings together with earlier data are taken as evidence that more than one type of heartwood formation exists.

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