Quantifying PaNAC03 Expression in Embryo Development

Samples for analysis of PaNAC03 expression levels during embryo development, was a generous gift from Drs. Irena Molina and Malin Abrahamsson. Briefly, samples were collected from five sequential developmental stages (classification based on Zhu et al. [35 (link)]): +PGR (Proliferating cultures + Plant growth regulators (PGR) five days after subculture), —PGR (Proliferating cultures —PGR five days after subculture), EE (Early embryos differentiated after one week on maturation medium); LE1 and LE2 (late early embryos developed after two and three weeks on maturation medium, respectively). Three independent samples were collected for every stage and frozen in liquid nitrogen and stored at −80 °C until extraction. Total RNA were extracted with the Spectrum Plant Total RNA kit (Sigma Aldrich) after DNAse I treatment one μg of total RNA was reverse transcribed with the Quanta cDNA synthesis kit (Quanta Biosciences).

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Dalman K., Wind J.J., Nemesio-Gorriz M., Hammerbacher A., Lundén K., Ezcurra I, & Elfstrand M. (2017). Overexpression of PaNAC03, a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development. BMC Plant Biology, 17, 6.

Publication 2017

Spectrum Plant Total RNA kit Quanta cDNA synthesis kit

Cdna Dnase Embryo development Embryos Frozen I rna Nitrogen Plant growth regulators Plant rna Rna i Synthesis

Corresponding Organization :

Other organizations : Swedish University of Agricultural Sciences, KTH Royal Institute of Technology, AlbaNova, Max Planck Institute for Chemical Ecology

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Variable analysis

independent variables

Developmental stages of embryo: +PGR (Proliferating cultures + Plant growth regulators (PGR) five days after subculture), —PGR (Proliferating cultures —PGR five days after subculture), EE (Early embryos differentiated after one week on maturation medium), LE1 (late early embryos developed after two weeks on maturation medium), and LE2 (late early embryos developed after three weeks on maturation medium)

dependent variables

Expression levels of PaNAC03 during embryo development

control variables

Three independent samples were collected for every developmental stage
Samples were frozen in liquid nitrogen and stored at −80 °C until extraction
Total RNA was extracted with the Spectrum Plant Total RNA kit (Sigma Aldrich) after DNAse I treatment
One μg of total RNA was reverse transcribed with the Quanta cDNA synthesis kit (Quanta Biosciences)

controls

No positive or negative controls were explicitly mentioned in the information provided.

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